bims-numges 2021-04-11 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–04–11
forty-four papers selected by
Sean Rudd, Karolinska Institutet

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.
Werner helicase is a synthetic-lethal vulnerability in Mismatch Repair-Deficient Colorectal Cancer Refractory to Targeted Therapies, Chemotherapy and Immunotherapy.
Enhanced cytarabine-induced killing in OGG1-deficient acute myeloid leukemia cells.
Targeting nucleotide metabolism enhances the efficacy of anthracyclines and anti-metabolites in triple-negative breast cancer.
The selection process of licensing a DNA mismatch for repair.
ARID1A regulates R-loop associated DNA replication stress.
Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide.
Which Holds the Key to BRCAness: Inability to Repair the Break, Protect the Fork, or Prevent the Gap?
A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity.
The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length.
DNA damage response and repair in osteosarcoma: Defects, regulation and therapeutic implications.
Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.
SHP2-mediated inhibition of DNA repair contributes to cGAS-STING activation and chemotherapeutic sensitivity in colon cancer.
Mitochondrial NADP+ is essential for proline biosynthesis during cell growth.
Repair of programmed DNA lesions in antibody class switch recombination: common and unique features.
Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.
Critical role of SMG7 in activation of the ATR-CHK1 axis in response to genotoxic stress.
Olaparib and rucaparib for the treatment of DNA repair-deficient metastatic castration-resistant prostate cancer.
Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites.
Mechanistic origins of diverse genome rearrangements in cancer.
Cell Metabolism and DNA Repair Pathways: Implications for Cancer Therapy.
R-loops as Janus-faced modulators of DNA repair.
Multinucleation associated DNA damage blocks proliferation in p53-compromised cells.
An 111In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells.
Metabotype analysis of Mthfd1l-null mouse embryos using desorption electrospray ionization mass spectrometry imaging.
Cyclin-dependent kinase inhibitors (CDKIs) and the DNA damage response: The link between signaling pathways and cancer.
Long-term survival of an ovarian cancer patient harboring a RAD51C missense mutation.
Inositol pyrophosphates promote MYC polyubiquitination by FBW7 to regulate cell survival.
Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia.
Fangchinoline suppresses conjunctival melanoma by directly binding FUBP2 and inhibiting the homologous recombination pathway.
Early nucleolar responses differentiate mechanisms of cell death induced by oxaliplatin and cisplatin.
Disruption of the MSL complex inhibits tumour maintenance by exacerbating chromosomal instability.
Integrating PARP Inhibitors Into Advanced Prostate Cancer Therapeutics.
Dynamic DNA Shortening by Telomere-Binding Protein Cdc13.
Clinical acquired resistance to KRASG12C inhibition through a novel KRAS switch-II pocket mutation and polyclonal alterations converging on RAS-MAPK reactivation.
CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer.
Discovery of AG-270, a First-in-Class Oral MAT2A Inhibitor for the Treatment of Tumors with Homozygous MTAP Deletion.
Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond.
A Very Long-Acting PARP Inhibitor Suppresses Cancer Cell Growth in DNA Repair-Deficient Tumor Models.
Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription.
Poly (ADP-ribose) polymerase inhibitors for arsenic trioxide-resistant acute promyelocytic leukemia: synergistic in vitro antitumor effects with hypomethylating agents or high-dose vitamin C.
The meiosis-specific MEIOB-SPATA22 complex cooperates with RPA to form a compacted mixed MEIOB/SPATA22/RPA/ssDNA complex.
Consequences of mitotic failure - The penalties and the rewards.
DNA molecular combing-based replication fork directionality profiling.

Science. 2021 Apr 09. 372(6538): 156-165

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.

Kasper Fugger, Ilirjana Bajrami, Mariana Silva Dos Santos, Sarah Jane Young, Simone Kunzelmann, Geoff Kelly, Graeme Hewitt, Harshil Patel, Robert Goldstone, Thomas Carell, Simon J Boulton, James MacRae, Ian A Taylor, Stephen C West.

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.

DOI: https://doi.org/10.1126/science.abb4542
Cancer Discov. 2021 Apr 09. pii: candisc.1508.2020. [Epub ahead of print]

Werner helicase is a synthetic-lethal vulnerability in Mismatch Repair-Deficient Colorectal Cancer Refractory to Targeted Therapies, Chemotherapy and Immunotherapy.

Gabriele Picco, Chiara Maria Cattaneo, Esmee J van Vliet, Giovanni Crisafulli, Giuseppe Rospo, Sarah Consonni, Sara Filipa Vieira, Inigo Sanchez Rodriguez, Carlotta Cancelliere, Ruby Banerjee, Luuk Johan Schipper, Daniele Oddo, Krijn Kristian Dijkstra, Jindrich Cinatl, Martin Michaelis, Fengtang Yang, Cell Model Network Uk Group, Federica Di Nicolantonio, Andrea Sartore-Bianchi, Salvatore Siena, Sabrina Arena, Emile E Voest, Alberto Bardelli, Mathew J Garnett.

Targeted therapies, chemotherapy, and immunotherapy are used to treat patients with mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer (CRC). The clinical effectiveness of targeted therapy and chemotherapy is limited by resistance and drug toxicities, and about half of immunotherapy patients are refractory to immune checkpoint inhibitors. Loss of Werner syndrome ATP-dependent helicase (WRN) is a synthetic-lethality in dMMR/MSI-H cells. To inform the development of WRN as a therapeutic target, we performed WRN knockout or knockdown in 60 heterogeneous dMMR CRC preclinical models, demonstrating that WRN dependency is an almost universal feature and a robust marker for patient selection. Furthermore, models of resistance to clinically relevant targeted therapy, chemotherapy, and immunotherapy retain WRN dependency. These data show the potential of therapeutically targeting WRN in dMMR/MSI-H CRC patients, and support WRN as a therapeutic option for patients with dMMR/MSI-H cancers refractory to current treatment strategies.

DOI: https://doi.org/10.1158/2159-8290.CD-20-1508
Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2016833118. [Epub ahead of print]118(11):

Enhanced cytarabine-induced killing in OGG1-deficient acute myeloid leukemia cells.

Nichole Owen, Irina G Minko, Samantha A Moellmer, Sydney K Cammann, R Stephen Lloyd, Amanda K McCullough.

  Human clinical trials suggest that inhibition of enzymes in the DNA base excision repair (BER) pathway, such as PARP1 and APE1, can be useful in anticancer strategies when combined with certain DNA-damaging agents or tumor-specific genetic deficiencies. There is also evidence suggesting that inhibition of the BER enzyme 8-oxoguanine DNA glycosylase-1 (OGG1), which initiates repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-dG), could be useful in treating certain cancers. Specifically, in acute myeloid leukemia (AML), both the RUNX1-RUNX1T1 fusion and the CBFB-MYH11 subtypes have lower levels of OGG1 expression, which correlate with increased therapeutic-induced cell cytotoxicity and good prognosis for improved, relapse-free survival compared with other AML patients. Here we present data demonstrating that AML cell lines deficient in OGG1 have enhanced sensitivity to cytarabine (cytosine arabinoside [Ara-C]) relative to OGG1-proficient cells. This enhanced cytotoxicity correlated with endogenous oxidatively-induced DNA damage and Ara-C-induced DNA strand breaks, with a large proportion of these breaks occurring at common fragile sites. This lethality was highly specific for Ara-C treatment of AML cells deficient in OGG1, with no other replication stress-inducing agents showing a correlation between cell killing and low OGG1 levels. The mechanism for this preferential toxicity was addressed using in vitro replication assays in which DNA polymerase δ was shown to insert Ara-C opposite 8-oxo-dG, resulting in termination of DNA synthesis. Overall, these data suggest that incorporation of Ara-C opposite unrepaired 8-oxo-dG may be the fundamental mechanism conferring selective toxicity and therapeutic effectiveness in OGG1-deficient AML cells.

Keywords:  AML therapy; DNA polymerase delta; DNA repair; DNA replication; fragile site

DOI:  https://doi.org/10.1073/pnas.2016833118
NPJ Breast Cancer. 2021 Apr 06. 7(1): 38

Targeting nucleotide metabolism enhances the efficacy of anthracyclines and anti-metabolites in triple-negative breast cancer.

Craig Davison, Roisin Morelli, Catherine Knowlson, Melanie McKechnie, Robbie Carson, Xanthi Stachtea, Kylie A McLaughlin, Vivien E Prise, Kienan Savage, Richard H Wilson, Karl A Mulligan, Peter M Wilson, Robert D Ladner, Melissa J LaBonte.

Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy.

DOI: https://doi.org/10.1038/s41523-021-00245-5
Nat Struct Mol Biol. 2021 Apr 05.

The selection process of licensing a DNA mismatch for repair.

Rafael Fernandez-Leiro, Doreth Bhairosing-Kok, Vladislav Kunetsky, Charlie Laffeber, Herrie H Winterwerp, Flora Groothuizen, Alexander Fish, Joyce H G Lebbink, Peter Friedhoff, Titia K Sixma, Meindert H Lamers.

DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.

DOI: https://doi.org/10.1038/s41594-021-00577-7
PLoS Genet. 2021 Apr 07. 17(4): e1009238

ARID1A regulates R-loop associated DNA replication stress.

Shuhe Tsai, Louis-Alexandre Fournier, Emily Yun-Chia Chang, James P Wells, Sean W Minaker, Yi Dan Zhu, Alan Ying-Hsu Wang, Yemin Wang, David G Huntsman, Peter C Stirling.

ARID1A is a core DNA-binding subunit of the BAF chromatin remodeling complex, and is lost in up to 7% of all cancers. The frequency of ARID1A loss increases in certain cancer types, such as clear cell ovarian carcinoma where ARID1A protein is lost in about 50% of cases. While the impact of ARID1A loss on the function of the BAF chromatin remodeling complexes is likely to drive oncogenic gene expression programs in specific contexts, ARID1A also binds genome stability regulators such as ATR and TOP2. Here we show that ARID1A loss leads to DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. These effects correlate with altered transcription and replication dynamics in ARID1A knockout cells and to reduced TOP2A binding at R-loop sites. Together this work extends mechanisms of replication stress in ARID1A deficient cells with implications for targeting ARID1A deficient cancers.

DOI: https://doi.org/10.1371/journal.pgen.1009238
Nat Commun. 2021 04 06. 12(1): 2059

Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide.

Joonas A Jamsen, Akira Sassa, David D Shock, William A Beard, Samuel H Wilson.

Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

DOI: https://doi.org/10.1038/s41467-021-21354-6
Cancer Res. 2021 Mar 01. 81(5): 1214-1215

Which Holds the Key to BRCAness: Inability to Repair the Break, Protect the Fork, or Prevent the Gap?

Christine E Canman.

Defects in genes crucial for the process of DNA repair by homology-directed DNA repair (HDR), such as BRCA1 and BRCA2, are well-known contributors to cancer pathogenesis as well as an Achilles' heel that can be exploited therapeutically. BRCA1/2-deficient cells are exquisitely sensitive to agents that stall replication forks, such as PARP inhibitors and platinating drugs, presumably due to the inability to repair double-stranded breaks that form as a consequence of replication fork collapse. BRCA1/2 also promote tolerance to DNA replication stress by protecting replication forks from nucleolytic degradation. Both biological endpoints involve the deposition of RAD51 onto single-stranded DNA (ssDNA) for homology searching and strand exchange during HDR repair, as well as protection of newly synthesized DNA from nucleolytic degradation (i.e., replication fork protection). In this issue of Cancer Research, Panzarino and colleagues performed multiple separation-of-function studies and identify the lesion most associated with intolerance to replication stress in BRCA1/2-deficient cells is persistent ssDNA gaps in newly synthesized DNA, resulting from a failure to restrain DNA replication. Mechanisms that suppress gap formation are closely associated with chemoresistance, and the authors' findings challenge the paradigm that lack of HR repair or fork protection underlie the phenotype known as BRCAness.See related article by Panzarino et al., p. 1388.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-4340
Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2016287118. [Epub ahead of print]118(11):

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity.

Aleksandar Zdravković, James M Daley, Arijit Dutta, Tatsuya Niwa, Yasuto Murayama, Shuji Kanamaru, Kentaro Ito, Takahisa Maki, Bilge Argunhan, Masayuki Takahashi, Hideo Tsubouchi, Patrick Sung, Hiroshi Iwasaki.

  The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

Keywords:  Ctp1/CtIP; Mre11-Rad50-Nbs1; double-strand break repair; fission yeast; homologous recombination

DOI:  https://doi.org/10.1073/pnas.2016287118
Nucleic Acids Res. 2021 Apr 06. pii: gkab232. [Epub ahead of print]

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length.

Dipti Vinayak Vernekar, Giordano Reginato, Céline Adam, Lepakshi Ranjha, Florent Dingli, Marie-Claude Marsolier, Damarys Loew, Raphaël Guérois, Bertrand Llorente, Petr Cejka, Valérie Borde.

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1's activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

DOI: https://doi.org/10.1093/nar/gkab232
DNA Repair (Amst). 2021 Mar 24. pii: S1568-7864(21)00061-6. [Epub ahead of print]102 103105

DNA damage response and repair in osteosarcoma: Defects, regulation and therapeutic implications.

Fatemeh Sadoughi, Parisa Maleki Dana, Zatollah Asemi, Bahman Yousefi.

  Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents which has the survival rate of 20% in its advanced stages. Osteosarcomas are mostly resistance to our common treatments. DNA damage response (DDR) is a specialized multistep process containing abundant proteins which are necessary for the survival of any cell and organism. DDR machinery detects a diversity of DNA lesions and inhibits the cell cycle progression if these lesions are not repairable. DDR is involved in aging, age-related diseases, and cancer. In recent years, DDR inhibitors have gained the attention of researches due to their potentials in offering novel therapeutic targets and improving the response of many cancers to either chemo- or radio-therapy. In this regard, we tried to gather a great body of evidence about the role of DDR ingredients in osteosarcoma's initiation/progression, prognosis, and treatment.

Keywords:  DNA damage response; Malignancy; Osteosarcoma; Radio-residence

DOI:  https://doi.org/10.1016/j.dnarep.2021.103105
Mol Cell. 2021 Apr 04. pii: S1097-2765(21)00214-8. [Epub ahead of print]

Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.

Alice Clare Newman, Mattia Falcone, Alejandro Huerta Uribe, Tong Zhang, Dimitris Athineos, Matthias Pietzke, Alexei Vazquez, Karen Blyth, Oliver David Kenneth Maddocks.

  Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

Keywords:  IDO1; IFNγ; PDAC; cancer immunology; cancer metabolism; epacadostat; formate; immunometabolism; immunotherapy; one-carbon metabolism; pancreas; serine; stellate cells; tryptophan; tumor microenvironment

DOI:  https://doi.org/10.1016/j.molcel.2021.03.019
Cancer Res. 2021 Apr 05. pii: canres.3738.2020. [Epub ahead of print]

SHP2-mediated inhibition of DNA repair contributes to cGAS-STING activation and chemotherapeutic sensitivity in colon cancer.

Bin Wei, Lingyan Xu, Wenjie Guo, Yuanyuan Wang, Jingjing Wu, Xiaofei Li, Xiaomin Cai, Jinbo Hu, Meijing Wang, Qiang Xu, Wen Liu, Yanhong Gu.

As a cytoplasmic sensor of double-stranded DNA (dsDNA), the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays an important role in anti-tumor immunity. In this study, we investigated the effect of Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) on tumor cell-intrinsic STING pathway activity and DNA repair in colon cancer. SHP2 interacted with and dephosphorylated poly ADP-ribose polymerase (PARP1) after DNA damage. PARP1 inhibition by SHP2 resulted in reduced DNA repair and accumulation of dsDNA in cells, thus promoting hyperactivation of the STING pathway. The SHP2 agonist lovastatin was able to enhance SHP2 activity and promote STING pathway activation. Moreover, lovastatin significantly enhanced the efficacy of chemotherapy in colon cancer models, in part via STING pathway-mediated anti-tumor immunity. These findings suggest that SHP2 exacerbates STING pathway activation by restricting PARP1-mediated DNA repair in tumor cells, providing a basis for the combined use of lovastatin and chemotherapy in the treatment of colon cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-3738
Nat Metab. 2021 Apr 08.

Mitochondrial NADP+ is essential for proline biosynthesis during cell growth.

Diem H Tran, Rushendhiran Kesavan, Halie Rion, Mona Hoseini Soflaee, Ashley Solmonson, Divya Bezwada, Hieu S Vu, Feng Cai, John A Phillips, Ralph J DeBerardinis, Gerta Hoxhaj.

Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.

DOI: https://doi.org/10.1038/s42255-021-00374-y
Genome Instab Dis. 2021 Mar 26. 1-11

Repair of programmed DNA lesions in antibody class switch recombination: common and unique features.

Yafang Shang, Fei-Long Meng.

  The adaptive immune system can diversify the antigen receptors to eliminate various pathogens through programmed DNA lesions at antigen receptor genes. In immune diversification, general DNA repair machineries are applied to transform the programmed DNA lesions into gene mutation or recombination events with common and unique features. Here we focus on antibody class switch recombination (CSR), and review the initiation of base damages, the conversion of damaged base to DNA double-strand break, and the ligation of broken ends. With an emphasis on the unique features in CSR, we discuss recent advances in the understanding of DNA repair/replication coordination, and ERCC6L2-mediated deletional recombination. We further elaborate the application of CSR in end-joining, resection and translesion synthesis assays. In the time of the COVID-19 pandemic, we hope it help to understand the generation of therapeutic antibodies.

Keywords:  AID; Class switch recombination; DNA repair; Deletional recombination; ERCC6L2; NHEJ; Rev7; Shieldin

DOI:  https://doi.org/10.1007/s42764-021-00035-0
DNA Repair (Amst). 2021 Mar 16. pii: S1568-7864(21)00056-2. [Epub ahead of print]102 103100

Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.

S Köcher, J Volquardsen, A Perugachi Heinsohn, C Petersen, D Roggenbuck, K Rothkamm, W Y Mansour.

  Analysis and quantification of residual, unrepaired DNA double-strand breaks by detecting damage-associated γH2AX or 53BP1 foci is a promising approach to evaluate radiosensitivity or radiosensitization in tumor cells. Manual foci quantification by eye is well-established but unsatisfactory due to inconsistent foci numbers between different observers, lack of information about foci size and intensity and the time-consuming scoring process. Therefore, automated foci counting is an important goal. Several software solutions for automated foci counting in separately acquired fluorescence microscopy images have been established. The AKLIDES NUK technology by Medipan combines automated microscopy and image processing/ counting, enabling affordable high throughput foci analysis as a routine application. Using this machine, automated foci counting is well established for lymphocytes but has not yet been reported for adherent tumor cells with their irregularly shaped nuclei and heterogeneous foci textures. Here we aimed to use the AKLIDES NUK system for adherent tumor cells growing in clusters. We identified cell separation as a critical step to ensure fast and reliable automated nuclei detection. We validated our protocol for the fully automated quantification of (i) the IR-dose dependent increase and (ii) the ATM as well as PARP inhibitor-induced radiosensitization. Collectively, with this protocol the AKLIDES NUK system facilitates cost effective, fast and high throughput quantitative fluorescence microscopic analysis of DNA damage induced foci such as γH2AX and 53BP1 in adherent tumor cells.

Keywords:  DNA double-strand breaks; Fully automated foci counting; Radiosensitivity; γH2AX / 53BP1 foci

DOI:  https://doi.org/10.1016/j.dnarep.2021.103100
Sci Rep. 2021 Apr 05. 11(1): 7502

Critical role of SMG7 in activation of the ATR-CHK1 axis in response to genotoxic stress.

Kathleen Ho, Hongwei Luo, Wei Zhu, Yi Tang.

CHK1 is a crucial DNA damage checkpoint kinase and its activation, which requires ATR and RAD17, leads to inhibition of DNA replication and cell cycle progression. Recently, we reported that SMG7 stabilizes and activates p53 to induce G1 arrest upon DNA damage; here we show that SMG7 plays a critical role in the activation of the ATR-CHK1 axis. Following genotoxic stress, SMG7-null cells exhibit deficient ATR signaling, indicated by the attenuated phosphorylation of CHK1 and RPA32, and importantly, unhindered DNA replication and fork progression. Through its 14-3-3 domain, SMG7 interacts directly with the Ser635-phosphorylated RAD17 and promotes chromatin retention of the 9-1-1 complex by the RAD17-RFC, an essential step to CHK1 activation. Furthermore, through maintenance of CHK1 activity, SMG7 controls G2-M transition and facilitates orderly cell cycle progression during recovery from replication stress. Taken together, our data reveals SMG7 as an indispensable signaling component in the ATR-CHK1 pathway during genotoxic stress response.

DOI: https://doi.org/10.1038/s41598-021-86957-x
Expert Opin Pharmacother. 2021 Apr 07. 1-8

Olaparib and rucaparib for the treatment of DNA repair-deficient metastatic castration-resistant prostate cancer.

Benjamin L Maughan, Emmanuel S Antonarakis.

   INTRODUCTION: Metastatic prostate cancer is a heterogeneous disease characterized by clinical and genomic heterogeneity. Many prostate cancers harbor mutations causing DNA repair deficiency, specifically homologous recombination deficiency, sensitizing to drugs that inhibit poly ADP-ribose polymerase (PARP). PARP is an enzyme that is involved in single-stranded DNA repair and is the target of newly approved treatments for metastatic prostate cancer.
AREAS COVERED: Here, the authors' review the clinical trials leading to the recent approvals of two PARP inhibitors (PARPi), olaparib and rucaparib, specifically TOPARP-A, TOPARP-B, PROfound and TRITON-2. They also compare the different FDA approvals for both of these medications and outline the safety of this class of drugs in prostate cancer.
EXPERT OPINION: Because PARPi are particularly effective in men with somatic or germline alterations in BRCA1 and BRCA2, we recommend that all men be tested for DNA alterations with next-generation sequencing in tumor cells obtained from either tissue or blood. We also recommend that olaparib or rucaparib be considered relatively early in the treatment sequence in metastatic castration-resistant prostate cancer patients with BRCA1 or BRCA2 mutations. Other DNA alterations might also sensitize to PARPi though the response rates are lower, so other standard therapies should be prioritized first.

Keywords:  Olaparib; metastatic castration-resistant prostate cancer; parp inhibitor; rucaparib

DOI:  https://doi.org/10.1080/14656566.2021.1912015
Nat Commun. 2021 04 08. 12(1): 2111

Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites.

Sumedha Agashe, Chinnu Rose Joseph, Teresa Anne Clarisse Reyes, Demis Menolfi, Michele Giannattasio, Anja Waizenegger, Barnabas Szakal, Dana Branzei.

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

DOI: https://doi.org/10.1038/s41467-021-22217-w
Semin Cell Dev Biol. 2021 Apr 03. pii: S1084-9521(21)00035-5. [Epub ahead of print]

Mechanistic origins of diverse genome rearrangements in cancer.

Rashmi Dahiya, Qing Hu, Peter Ly.

  Cancer genomes frequently harbor structural chromosomal rearrangements that disrupt the linear DNA sequence order and copy number. To date, diverse classes of structural variants have been identified across multiple cancer types. These aberrations span a wide spectrum of complexity, ranging from simple translocations to intricate patterns of rearrangements involving multiple chromosomes. Although most somatic rearrangements are acquired gradually throughout tumorigenesis, recent interrogation of cancer genomes have uncovered novel categories of complex rearrangements that arises rapidly through a one-off catastrophic event, including chromothripsis and chromoplexy. Here we review the cellular and molecular mechanisms contributing to the formation of diverse structural rearrangement classes during cancer development. Genotoxic stress from a myriad of extrinsic and intrinsic sources can trigger DNA double-strand breaks that are subjected to DNA repair with potentially mutagenic outcomes. We also highlight how aberrant nuclear structures generated through mitotic cell division errors, such as rupture-prone micronuclei and chromosome bridges, can instigate massive DNA damage and the formation of complex rearrangements in cancer genomes.

Keywords:  Cancer genomes; Chromosome rearrangements; Chromothripsis; DNA damage; DNA repair; Genomic instability; Micronuclei; Mitosis

DOI:  https://doi.org/10.1016/j.semcdb.2021.03.003
Front Cell Dev Biol. 2021 ;9 633305

Cell Metabolism and DNA Repair Pathways: Implications for Cancer Therapy.

Thais Sobanski, Maddison Rose, Amila Suraweera, Kenneth O'Byrne, Derek J Richard, Emma Bolderson.

  DNA repair and metabolic pathways are vital to maintain cellular homeostasis in normal human cells. Both of these pathways, however, undergo extensive changes during tumorigenesis, including modifications that promote rapid growth, genetic heterogeneity, and survival. While these two areas of research have remained relatively distinct, there is growing evidence that the pathways are interdependent and intrinsically linked. Therapeutic interventions that target metabolism or DNA repair systems have entered clinical practice in recent years, highlighting the potential of targeting these pathways in cancer. Further exploration of the links between metabolic and DNA repair pathways may open new therapeutic avenues in the future. Here, we discuss the dependence of DNA repair processes upon cellular metabolism; including the production of nucleotides required for repair, the necessity of metabolic pathways for the chromatin remodeling required for DNA repair, and the ways in which metabolism itself can induce and prevent DNA damage. We will also discuss the roles of metabolic proteins in DNA repair and, conversely, how DNA repair proteins can impact upon cell metabolism. Finally, we will discuss how further research may open therapeutic avenues in the treatment of cancer.

Keywords:  DNA repair; cell metabolism; glycolysis; homologous recombination; non-homologous end-joining; tumor metabolic reprogramming; warburg effect

DOI:  https://doi.org/10.3389/fcell.2021.633305
Nat Cell Biol. 2021 Apr;23(4): 305-313

R-loops as Janus-faced modulators of DNA repair.

Aline Marnef, Gaëlle Legube.

R-loops are non-B DNA structures with intriguing dual consequences for gene expression and genome stability. In addition to their recognized roles in triggering DNA double-strand breaks (DSBs), R-loops have recently been demonstrated to accumulate in cis to DSBs, especially those induced in transcriptionally active loci. In this Review, we discuss whether R-loops actively participate in DSB repair or are detrimental by-products that must be removed to avoid genome instability.

DOI: https://doi.org/10.1038/s41556-021-00663-4
Commun Biol. 2021 Apr 09. 4(1): 451

Multinucleation associated DNA damage blocks proliferation in p53-compromised cells.

Madeleine Hart, Sophie D Adams, Viji M Draviam.

Nuclear atypia is one of the hallmarks of cancers. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of nuclear atypia. Tracking the fate of newborn cells exhibiting nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because ~50% of cancers display compromised p53, we explored how multinucleation blocks proliferation. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Multinucleation Associated DNA Damage associated with 53BP1-bodies remains unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. Persistent DNA damage, a DNA replication block, and reduced phospho-Rb, reveal a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

DOI: https://doi.org/10.1038/s42003-021-01979-5
Chem Sci. 2020 Sep 07. 11(33): 8936-8944

An 111In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells.

Martin R Gill, Michael G Walker, Sarah Able, Ole Tietz, Abirami Lakshminarayanan, Rachel Anderson, Rod Chalk, Afaf H El-Sagheer, Tom Brown, Jim A Thomas, Katherine A Vallis.

Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics. Herein we report the synthesis of a mismatch DNA binding bis-ruthenium(ii) dipyridophenazine (dppz) complex that can be radiolabelled with the Auger electron emitting radionuclide indium-111 (111In). Greater stabilisation accompanied by enhanced MLCT (metal to ligand charge-transfer) luminescence of both the bis-Ru(dppz) chelator and non-radioactive indium-loaded complex was observed in the presence of a TT mismatch-containing duplex compared to matched DNA. The radioactive construct [111In]In-bisRu(dppz) ([111In][In-2]4+) targets cell nuclei and is radiotoxic towards MMR-deficient human colorectal cancer cells showing substantially less detrimental effects in a paired cell line with restored MMR function. Additional cell line studies revealed that [111In][In-2]4+ is preferentially radiotoxic towards MMR-deficient colorectal cancer cells accompanied by increased DNA damage due to 111In decay. The biodistribution of [111In][In-2]4+ in live mice was demonstrated using SPECT. These results illustrate how a Ru(ii) polypyridyl complex can incorporate mismatch DNA binding and radiometal chelation in a single molecule, generating a DNA-targeting AE radiopharmaceutical that displays selective radiotoxicity towards MMR-deficient cancer cells and is compatible with whole organism SPECT imaging.

DOI: https://doi.org/10.1039/d0sc02825h
Anal Bioanal Chem. 2021 Apr 08.

Metabotype analysis of Mthfd1l-null mouse embryos using desorption electrospray ionization mass spectrometry imaging.

Amanda Vaughn, Rachel J DeHoog, Livia S Eberlin, Dean R Appling.

  Mammalian folate-dependent one-carbon (1C) metabolism provides the building blocks essential during development via amino acid interconversion, methyl-donor production, regeneration of redox factors, and de novo purine and thymidylate synthesis. Folate supplementation prevents many neural tube defects (NTDs) that occur during the embryonic process of neurulation. The mechanism by which folate functions during neurulation is not well understood, and not all NTDs are preventable by folate supplementation. Mthfd1l is a mitochondrial 1C metabolism enzyme that produces formate, a 1C donor that fuels biosynthesis and the methyl cycle in the cytoplasm. Homozygous deletion of the Mthfd1l gene in mice (Mthfd1lz/z) causes embryonic lethality, developmental delay, and folate-resistant NTDs. These mice also have defects in cranial mesenchyme formation. In this work, mass spectrometry imaging was used to obtain ion maps of the cranial mesenchyme that identified the spatial distribution and relative abundance of metabolites in wild-type and Mthfd1lz/z embryos. The relative abundances of purine and thymidylate derivatives, as well as amino acids, were diminished in the cranial mesenchyme of Mthfd1lz/z embryos. Loss of Mthfd1l activity in this region also led to abnormal levels of methionine and dysregulated energy metabolism. These alterations in metabolism suggest possible approaches to preventing NTDs in humans.

Keywords:  DESI; Folate; Imaging mass spectrometry; Metabolomics; Mitochondrial metabolism; Mthfd1l

DOI:  https://doi.org/10.1007/s00216-021-03308-5
DNA Repair (Amst). 2021 Mar 26. pii: S1568-7864(21)00059-8. [Epub ahead of print]102 103103

Cyclin-dependent kinase inhibitors (CDKIs) and the DNA damage response: The link between signaling pathways and cancer.

Jafar Amani, Nassim Gorjizadeh, Simin Younesi, Mojtaba Najafi, Arash M Ashrafi, Saeed Irian, Negar Gorjizadeh, Khalil Azizian.

  At the cellular level, DNA repair mechanisms are crucial in maintaining both genomic integrity and stability. DNA damage appears to be a central culprit in tumor onset and progression. Cyclin-dependent kinases (CDKs) and their regulatory partners coordinate the cell cycle progression. Aberrant CDK activity has been linked to a variety of cancers through deregulation of cell-cycle control. Besides DNA damaging agents and chromosome instability (CIN), disruptions in the levels of cell cycle regulators including cyclin-dependent kinase inhibitors (CDKIs) would result in unscheduled proliferation and cell division. The INK4 and Cip/Kip (CDK interacting protein/kinase inhibitor protein) family of CDKI proteins are involved in cell cycle regulation, transcription regulation, apoptosis, and cell migration. A thorough understanding of how these CDKIs regulate the DNA damage response through multiple signaling pathways may provide an opportunity to design efficient treatment strategies to inhibit carcinogenesis.

Keywords:  Cell cycle; Cyclin-dependent kinase inhibitors; DNA damage response

DOI:  https://doi.org/10.1016/j.dnarep.2021.103103
Cold Spring Harb Mol Case Stud. 2021 Apr;pii: a006083. [Epub ahead of print]7(2):

Long-term survival of an ovarian cancer patient harboring a RAD51C missense mutation.

Meghan R Sullivan, Rohit Prakash, Yashpal Rawal, Weibin Wang, Patrick Sung, Marc R Radke, Scott H Kaufmann, Elizabeth M Swisher, Kara A Bernstein, Maria Jasin.

  Mutations in homologous recombination (HR) genes predispose to cancer but also sensitize to chemotherapeutics. Although therapy can initially be effective, cancers frequently cease responding, leading to recurrence and poor prognosis. Here we identify a germline mutation in RAD51C, a critical HR factor and known tumor suppressor, in an ovarian cancer patient with exceptionally long, progression-free survival. The RAD51C-T132P mutation is in a highly conserved residue within the nucleotide-binding site and interferes with single-strand DNA binding of the RAD51 paralog complex RAD51B-RAD51C-RAD51D-XRCC2 and association with another RAD51 paralog XRCC3. These biochemical defects lead to highly defective HR and drug sensitivity in tumor cells, ascribing RAD51C-T132P as a deleterious mutation that was likely causal for tumor formation. Conversely, its position within a critical site suggests that it is refractory to secondary mutations that would restore RAD51C gene function and lead to therapy resistance. A need for a greater understanding of the relationship between mutation position and reversion potential of HR genes is underscored, as it may help predict the effectiveness of therapies in patients with HR-deficient cancers.

Keywords:  ovarian neoplasm

DOI:  https://doi.org/10.1101/mcs.a006083
Biochem J. 2021 Apr 06. pii: BCJ20210081. [Epub ahead of print]

Inositol pyrophosphates promote MYC polyubiquitination by FBW7 to regulate cell survival.

Padmavathi Lolla, Akruti Shah, Unnikannan C P, Vineesha Oddi, Rashna Bhandari.

  The transcription factor MYC regulates cell survival and growth, and its level is tightly controlled in normal cells. We report that serine pyrophosphorylation - a posttranslational modification triggered by inositol pyrophosphate signaling molecules - controls MYC levels via regulated protein degradation. We find that endogenous MYC is stabilized and less polyubiquitinated in cells with reduced inositol pyrophosphates. We show that the inositol pyrophosphate 5-IP7 transfers its high-energy beta phosphate moiety to pre-phosphorylated serine residues in the central PEST domain of MYC. Loss of serine pyrophosphorylation in the PEST domain lowers the extent of MYC polyubiquitination and increases its stability. Fusion to the MYC PEST domain lowers the stability of GFP, but this effect is dependent on the extent of PEST domain pyrophosphorylation. The E3 ubiquitin ligase FBW7 can bind directly to the PEST domain of MYC, and this interaction is exclusively dependent on serine pyrophosphorylation. A stabilized, pyrophosphorylation-deficient form of MYC increases cell death during growth stress in untransformed cells. Splenocytes from mice lacking IP6K1, a kinase responsible for the synthesis of 5-IP7, have higher levels of MYC, and show increased cell proliferation in response to mitogens, compared with splenocytes from wild type mice. Thus, control of MYC stability through a novel pyro-phosphodegron provides unexpected insight into the regulation of cell survival in response to environmental cues.

Keywords:  IP7; inositol hexakisphosphate kinase; inositol pyrophosphates; serine pyrophosphorylation

DOI:  https://doi.org/10.1042/BCJ20210081
Nat Genet. 2021 Apr 08.

Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia.

Zhimin Gu, Yuxuan Liu, Yuannyu Zhang, Hui Cao, Junhua Lyu, Xun Wang, Annika Wylie, Simon J Newkirk, Amanda E Jones, Michael Lee, Giovanni A Botten, Mi Deng, Kathryn E Dickerson, Cheng Cheng Zhang, Wenfeng An, John M Abrams, Jian Xu.

Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.

DOI: https://doi.org/10.1038/s41588-021-00829-8
Cell Death Dis. 2021 Apr 07. 12(4): 380

Fangchinoline suppresses conjunctival melanoma by directly binding FUBP2 and inhibiting the homologous recombination pathway.

Keting Bao, Yongyun Li, Jinlian Wei, Ruoxi Li, Jie Yang, Jiahao Shi, Baoli Li, Jin Zhu, Fei Mao, Renbing Jia, Jian Li.

Conjunctival melanoma (CM) is a rare and fatal ocular tumour with poor prognosis. There is an urgent need of effective therapeutic drugs against CM. Here, we reported the discovery of a novel potential therapeutic target for CM. Through phenotypic screening of our in-house library, fangchinoline was discovered to significantly inhibit the growth of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1. Further mechanistic experiments indicated that fangchinoline suppressed the homologous recombination (HR)-directed DNA repair by binding with far upstream element binding protein 2 (FUBP2) and downregulating the expression of HR factors BRCA1 and RAD51. In vitro and in vivo antitumour experiments revealed that fangchinoline increased the efficacy of cisplatin by blocking HR factors and reduced the drug dose and toxicity. In conclusion, our work provides a promising therapeutic strategy for the treatment of CM that is worthy of extensive preclinical investigation.

DOI: https://doi.org/10.1038/s41419-021-03653-4
J Biol Chem. 2021 Apr 02. pii: S0021-9258(21)00419-1. [Epub ahead of print] 100633

Early nucleolar responses differentiate mechanisms of cell death induced by oxaliplatin and cisplatin.

Emily C Sutton, Victoria J DeRose.

  Recent reports provide evidence that the platinum chemotherapeutic oxaliplatin causes cell death via ribosome biogenesis stress, while cisplatin causes cell death via the DNA damage response (DDR). Underlying differences in mechanisms that might initiate disparate routes to cell death by these two broadly used platinum compounds have not yet been carefully explored. Additionally, prior studies had demonstrated that cisplatin can also inhibit ribosome biogenesis. Therefore, we sought to directly compare the initial influences of oxaliplatin and cisplatin on nucleolar processes and on the DDR. Using pulse-chase experiments, we found that at equivalent doses, oxaliplatin but not cisplatin significantly inhibited ribosomal RNA (rRNA) synthesis by Pol I, but neither compound affected rRNA processing. Inhibition of rRNA synthesis occurred as early as 90 minutes after oxaliplatin treatment in A549 cells, concurrent with the initial redistribution of the nucleolar protein nucleophosmin (NPM1). We observed that the nucleolar protein fibrillarin began to redistribute by 6 hours after oxaliplatin treatment, and formed canonical nucleolar caps by 24 hours. In cisplatin-treated cells, DNA damage, as measured by γH2AX immunofluorescence, was more extensive, whereas nucleolar organization was unaffected. Taken together, our results demonstrate that oxaliplatin causes early nucleolar disruption via inhibition of rRNA synthesis accompanied by NPM1 relocalization, and subsequently causes extensive nucleolar reorganization, while cisplatin causes early DNA damage without significant nucleolar disruption. These data support a model in which, at clinically relevant doses, cisplatin kills cells via the canonical DDR, and oxaliplatin kills cells via ribosome biogenesis stress, specifically via rapid inhibition of rRNA synthesis.

Keywords:  DNA damage response; Nucleolus; anticancer drug; nucleolar stress; pre-rRNA; ribosomal RNA; ribosome biogenesis; stress response

DOI:  https://doi.org/10.1016/j.jbc.2021.100633
Nat Cell Biol. 2021 Apr;23(4): 401-412

Disruption of the MSL complex inhibits tumour maintenance by exacerbating chromosomal instability.

Josep Monserrat, Cristina Morales Torres, Louise Richardson, Thomas Stuart Wilson, Harshil Patel, Marie-Charlotte Domart, Stuart Horswell, Ok-Ryul Song, Ming Jiang, Margaret Crawford, Minh Bui, Yamini Dalal, Paola Scaffidi.

Rewiring of cellular programmes in malignant cells generates cancer-specific vulnerabilities. Here, using an unbiased screening strategy aimed at identifying non-essential genes required by tumour cells to sustain unlimited proliferative capacity, we identify the male-specific lethal (MSL) acetyltransferase complex as a vulnerability of genetically unstable cancers. We find that disruption of the MSL complex and consequent loss of the associated H4K16ac mark do not substantially alter transcriptional programmes but compromise chromosome integrity and promote chromosomal instability (CIN) that progressively exhausts the proliferative potential of cancer cells through a p53-independent mechanism. This effect is dependent on pre-existing genomic instability, and normal cells are insensitive to MSL disruption. Using cell- and patient-derived xenografts from multiple cancer types, we show that excessive CIN induced by MSL disruption inhibits tumour maintenance. Our findings suggest that targeting MSL may be a valuable means to increase CIN beyond the level tolerated by cancer cells without inducing severe adverse effects in normal tissues.

DOI: https://doi.org/10.1038/s41556-021-00657-2
Oncology (Williston Park). 2021 Mar 15. 35(3): 119-125

Integrating PARP Inhibitors Into Advanced Prostate Cancer Therapeutics.

Jun Gong, Edwin Posadas, Neil Bhowmick, Hyung Kim, Timothy Daskivich, Amit Gupta, Howard Sandler, Mitchell Kamrava, Zachary Zumsteg, Stephen Freedland, Robert Figlin.

  DNA-damage repair (DDR) pathway mutations can sensitize cancer cells to a class of cancer therapeutics known as PARP inhibitors. Given that DDR alterations can be found in up to one-third of advanced prostate cancers, PARP inhibitors have recently been established in treatment-refractory settings. We provide an updated review of the clinical data supporting the 4 PARP inhibitors that have undergone the most investigation thus far in metastatic castrate-resistant prostate cancer (mCRPC). Two of these agents are currently approved for the treatment of DDR-altered mCRPC. We end with a discussion on integration of approved PARP inhibitors into advanced prostate cancer clinical practice.

Keywords:  BRCA; DNA damage repair; PARP inhibitors; androgen inhibition; homologous recombination; poly(ADP) ribose polymerase; prostate cancer

DOI:  https://doi.org/10.46883/ONC.2021.3503.0119
J Am Chem Soc. 2021 Apr 08.

Dynamic DNA Shortening by Telomere-Binding Protein Cdc13.

Yi-Yun Lin, Min-Hsuan Li, Yen-Chan Chang, Peng-Yu Fu, Ryosuke L Ohniwa, Hung-Wen Li, Jing-Jer Lin.

Telomeres are essential for chromosome maintenance. Cdc13 is a single-stranded telomeric DNA binding protein that caps telomeres and regulates telomerase function in yeast. Although specific binding of Cdc13 to telomeric DNA is critical for telomere protection, the detail mechanism how Cdc13-DNA complex protects telomere is unclear. Using two single-molecule methods, tethered particle motion and atomic force microscopy, we demonstrate that specific binding of Cdc13 on single-stranded telomeric DNA shortens duplex DNA into distinct states differed by ∼70-80 base pairs. DNA shortening by Cdc13 is dynamic and independent of duplex DNA sequences or length. Significantly, we found that Pif1 helicase is incapable of removing Cdc13 from the shortened DNA-Cdc13 complex, suggesting that Cdc13 forms structurally stable complex by shortening of the bound DNA. Together our data identified shortening of DNA by Cdc13 and provided an indication for efficient protection of telomere ends by the shortened DNA-Cdc13 complex.

DOI: https://doi.org/10.1021/jacs.1c00820
Cancer Discov. 2021 Apr 06. pii: candisc.0365.2021. [Epub ahead of print]

Clinical acquired resistance to KRASG12C inhibition through a novel KRAS switch-II pocket mutation and polyclonal alterations converging on RAS-MAPK reactivation.

Noritaka Tanaka, Jessica J Lin, Chendi Li, Meagan B Ryan, Junbing Zhang, Lesli A Kiedrowski, Alexa G Michel, Mohammed U Syed, Katerina A Fella, Mustafa Sakhi, Islam Baiev, Dejan Juric, Justin F Gainor, Samuel J Klempner, Jochen K Lennerz, Giulia Siravegna, Liron Bar-Peled, Aaron N Hata, Rebecca S Heist, Ryan B Corcoran.

Mutant-selective KRASG12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRASG12C-mutant cancers including non-small cell lung cancer (NSCLC). However, mechanisms underlying clinical acquired resistance to KRASG12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a KRASG12C NSCLC patient who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS-MAPK signaling. Notably, a novel KRASY96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein-drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRASG12C cancer models. Interestingly, a novel, functionally distinct tri-complex KRASG12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRASG12C/Y96D and could overcome resistance.

DOI: https://doi.org/10.1158/2159-8290.CD-21-0365
Front Oncol. 2021 ;11 637470

CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer.

Huaizhu Chu, Nahui Han, Jianguo Xu.

  Gastric cancer (GC) remains a major world-wide challenge, especially in Asian countries. Chemotherapy with 5-fluorouracil (5-FU) and cisplatin is used as the first-line treatment and development of chemoresistance is a major cause of progression. UMP/CMP kinase is responsible for the phosphorylation of the ribonucleotide metabolite 5-fluoro-5'-monophosphate (FUMP) in 5-FU metabolic process, and recognized as a key step in the conversion of 5-FU to cytotoxic metabolites. Our bioinformatics analysis and molecular experiments demonstrated that high expression of CMPK1 was associated with prolonged survival and response to 5-FU treatment in GC samples. Further analysis demonstrated that miR-130b as a key epigenetic regulator of CMPK1, and miR-130b-mediated attenuation of CMPK1 resulted in resistance of gastric cancer cells to DNA damage and cell death after treatment with 5-FU. Rescue experiments with augmented CMPK1 expression abolished the effect of miR-130b demonstrating the key function of this miRNA in this pathway. Thus, this newly identified miR-130b-CMPK1 axis suggests a potentially new chemotherapeutic strategy for improved response to 5-FU therapy.

Keywords:  5-FU; CMPK; MiR-130b; chemoresistance; gastric cancer

DOI:  https://doi.org/10.3389/fonc.2021.637470
J Med Chem. 2021 Apr 08.

Discovery of AG-270, a First-in-Class Oral MAT2A Inhibitor for the Treatment of Tumors with Homozygous MTAP Deletion.

Zenon Konteatis, Jeremy Travins, Stefan Gross, Katya Marjon, Amelia Barnett, Everton Mandley, Brandon Nicolay, Raj Nagaraja, Yue Chen, Yabo Sun, Zhixiao Liu, Jie Yu, Zhixiong Ye, Fan Jiang, Wentao Wei, Cheng Fang, Yi Gao, Peter Kalev, Marc L Hyer, Byron DeLaBarre, Lei Jin, Anil K Padyana, Lenny Dang, Joshua Murtie, Scott A Biller, Zhihua Sui, Kevin M Marks.

The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10 000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).

DOI: https://doi.org/10.1021/acs.jmedchem.0c01895
Front Oncol. 2021 ;11 612354

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond.

Michael Orth, Valerie Albrecht, Karin Seidl, Linda Kinzel, Kristian Unger, Julia Hess, Lisa Kreutzer, Na Sun, Benjamin Stegen, Alexander Nieto, Jessica Maas, Nicolas Winssinger, Anna A Friedl, Axel K Walch, Claus Belka, Horst Zitzelsberger, Maximilian Niyazi, Kirsten Lauber.

  Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

Keywords:  HSP90 inhibition; HSP90i; NW457; glioblastoma; hypermigration; radiosensitization; radiotherapy

DOI:  https://doi.org/10.3389/fonc.2021.612354
Cancer Res. 2021 Feb 15. 81(4): 1076-1086

A Very Long-Acting PARP Inhibitor Suppresses Cancer Cell Growth in DNA Repair-Deficient Tumor Models.

Shaun D Fontaine, Gary W Ashley, Peter J Houghton, Raushan T Kurmasheva, Morgan Diolaiti, Alan Ashworth, Cody J Peer, Ryan Nguyen, William D Figg, Denis R Beckford-Vera, Daniel V Santi.

PARP inhibitors are approved for treatment of cancers with BRCA1 or BRCA2 defects. In this study, we prepared and characterized a very long-acting PARP inhibitor. Synthesis of a macromolecular prodrug of talazoparib (TLZ) was achieved by covalent conjugation to a PEG40kDa carrier via a β-eliminative releasable linker. A single injection of the PEG∼TLZ conjugate was as effective as ∼30 daily oral doses of TLZ in growth suppression of homologous recombination-defective tumors in mouse xenografts. These included the KT-10 Wilms' tumor with a PALB2 mutation, the BRCA1-deficient MX-1 triple-negative breast cancer, and the BRCA2-deficient DLD-1 colon cancer; the prodrug did not inhibit an isogenic DLD-1 tumor with wild-type BRCA2. Although the half-life of PEG∼TLZ and released TLZ in the mouse was only ∼1 day, the exposure of released TLZ from a single safe, effective dose of the prodrug exceeded that of oral TLZ given daily over one month. μPET/CT imaging showed high uptake and prolonged retention of an 89Zr-labeled surrogate of PEG∼TLZ in the MX-1 BRCA1-deficient tumor. These data suggest that the long-lasting antitumor effect of the prodrug is due to a combination of its long t 1/2, the high exposure of TLZ released from the prodrug, increased tumor sensitivity upon continued exposure, and tumor accumulation. Using pharmacokinetic parameters of TLZ in humans, we designed a long-acting PEG∼TLZ for humans that may be superior in efficacy to daily oral TLZ and would be useful for treatment of PARP inhibitor-sensitive cancers in which oral medications are not tolerated. SIGNIFICANCE: These findings demonstrate that a single injection of a long-acting prodrug of the PARP inhibitor talazoparib in murine xenografts provides tumor suppression equivalent to a month of daily dosing of talazoparib.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-1741
Cancer Cell. 2021 Mar 30. pii: S1535-6108(21)00164-1. [Epub ahead of print]

Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription.

Yanfen Zhu, Amit D Gujar, Chee-Hong Wong, Harianto Tjong, Chew Yee Ngan, Liang Gong, Yi-An Chen, Hoon Kim, Jihe Liu, Meihong Li, Adam Mil-Homens, Rahul Maurya, Chris Kuhlberg, Fanyue Sun, Eunhee Yi, Ana C deCarvalho, Yijun Ruan, Roel G W Verhaak, Chia-Lin Wei.

  Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.

Keywords:  ChIA-Drop; ChIA-PET; chromatin interactions; ecDNA; mobile enhancers

DOI:  https://doi.org/10.1016/j.ccell.2021.03.006
J Pharmacol Exp Ther. 2021 Apr 05. pii: JPET-AR-2021-000537. [Epub ahead of print]

Poly (ADP-ribose) polymerase inhibitors for arsenic trioxide-resistant acute promyelocytic leukemia: synergistic in vitro antitumor effects with hypomethylating agents or high-dose vitamin C.

Manuela Giansanti, Antonio De Gabrieli, Salvatore Pasquale Prete, Tiziana Ottone, Maria Domenica Divona, Terry Karimi, Fabio Ciccarone, Maria Teresa Voso, Grazia Graziani, Isabella Faraoni.

  Arsenic trioxide (ATO) is an anticancer agent used for the treatment of acute promyelocytic leukemia (APL). However, 5-10% of patients fail to respond or experience disease relapse. Based on poly(ADP-ribose) polymerase 1 (PARP1) involvement in the processing of DNA demethylation, here we have tested the in vitro susceptibility of ATO-resistant clones, derived from the human APL cell line NB4, to PARP inhibitors (PARPi) in combination with hypomethylating agents (azacitidine and decitabine) or high-dose vitamin C (ascorbate), which induces 5-hydroxymethylcytosine (5hmC)-mediated DNA demethylation. ATO-sensitive and -resistant APL cell clones were generated and initially analyzed for their susceptibility to five clinically used PARPi (olaparib, niraparib, rucaparib, veliparib and talazoparib). The obtained PARPi IC50 values were far below (olaparib and niraparib), within the range (talazoparib) or above (rucaparib and veliparib) the Cmax reported in patients, likely due to differences in the mechanisms of their cytotoxic activity. ATO-resistant APL cells were also susceptible to clinically relevant concentrations of azacitidine and decitabine and to high-dose ascorbate. Interestingly, the combination of these agents with olaparib, niraparib or talazoparib resulted in synergistic antitumor activity. In combination with ascorbate, PARPi increased the ascorbate-mediated induction of 5hmC, which likely resulted in stalled DNA repair and cytotoxicity. Talazoparib was the most effective PARPi in synergizing with ascorbate, in accordance with its marked ability to trap PARP1 at damaged DNA. These findings suggest that ATO and PARPi have non-overlapping resistance mechanisms and support further investigation on PARPi combination with hypomethylating agents or high-dose ascorbate for relapsed/ATO-refractory APL especially in frail patients. Significance Statement In this study we found that poly(ADP-ribose) inhibitors (PARPi) show activity as single agents against human acute promyelocytic leukemia cells resistant to arsenic trioxide at clinical relevant concentrations. Furthermore, PARPi enhance the in vitro efficacy of high-dose vitamin C, azacitidine and decitabine, all agents that alter DNA methylation. In combination with vitamin C, PARPi increase the levels of 5-hydroxymethylcytosine, likely because of altered processing of the oxidized intermediates associated with DNA demethylation.

Keywords:  DNA damage; leukemia

DOI:  https://doi.org/10.1124/jpet.121.000537
DNA Repair (Amst). 2021 Mar 13. pii: S1568-7864(21)00053-7. [Epub ahead of print]102 103097

The meiosis-specific MEIOB-SPATA22 complex cooperates with RPA to form a compacted mixed MEIOB/SPATA22/RPA/ssDNA complex.

Jonathan Ribeiro, Pauline Dupaigne, Cynthia Petrillo, Cécile Ducrot, Clotilde Duquenne, Xavier Veaute, Carole Saintomé, Didier Busso, Raphaël Guerois, Emmanuelle Martini, Gabriel Livera.

  During meiosis, programmed double-strand breaks are repaired by homologous recombination (HR) to form crossovers that are essential to homologous chromosome segregation. Single-stranded DNA (ssDNA) containing intermediates are key features of HR, which must be highly regulated. RPA, the ubiquitous ssDNA binding complex, was thought to play similar roles during mitotic and meiotic HR until the recent discovery of MEIOB and its partner, SPATA22, two essential meiosis-specific proteins. Here, we show that like MEIOB, SPATA22 resembles RPA subunits and binds ssDNA. We studied the physical and functional interactions existing between MEIOB, SPATA22, and RPA, and show that MEIOB and SPATA22 interact with the preformed RPA complex through their interacting domain and condense RPA-coated ssDNA in vitro. In meiotic cells, we show that MEIOB and SPATA22 modify the immunodetection of the two large subunits of RPA. Given these results, we propose that MEIOB-SPATA22 and RPA form a functional ssDNA-interacting complex to satisfy meiotic HR requirements by providing specific properties to the ssDNA.

Keywords:  Gametogenesis; Meiosis; Meiotic recombination; OB domain

DOI:  https://doi.org/10.1016/j.dnarep.2021.103097
Semin Cell Dev Biol. 2021 Apr 02. pii: S1084-9521(21)00039-2. [Epub ahead of print]

Consequences of mitotic failure - The penalties and the rewards.

Zuzana Storchova.

  Eukaryotic cells are usually diploid, meaning they contain two copies of each chromosome. However, aberrant chromosome numbers due to both, chromosome gains and losses, are often observed in nature. They can occur as a planned developmental step, but are more often an uninvited result of mitotic failure. Recent discoveries have improved our understanding of the cellular effects of aneuploidy - uneven chromosome numbers, and polyploidy - multiplication of entire sets of chromosomes - in eukaryotic cells. The results show that mitotic errors lead to rapid and extensive modifications of many cellular processes and affect proliferation, proteome balance, genome stability and more. The findings picture the cellular response to aneuploidy and polyploidy as a complex, tissue and context dependent network of events. Here I review the latest discoveries, with an emphasis on pathological aspects of aneuploidy and polyploidy in human cells.

Keywords:  Aneuploidy; Cancer; Chromosomal instability; Chromosome missegregation; Chromothripsis; Mitosis

DOI:  https://doi.org/10.1016/j.semcdb.2021.03.007
Nucleic Acids Res. 2021 Apr 09. pii: gkab219. [Epub ahead of print]

DNA molecular combing-based replication fork directionality profiling.

Marion Blin, Laurent Lacroix, Nataliya Petryk, Yan Jaszczyszyn, Chun-Long Chen, Olivier Hyrien, Benoît Le Tallec.

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

DOI: https://doi.org/10.1093/nar/gkab219