UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2020

(2020 Oct 1)

Flavaglines as natural products targeting eIF4A and prohibitins: From traditional Chinese medicine to antiviral activity against coronaviruses

Eur J Med Chem. : DOI : 10.1016/j.ejmech.2020.112653 En savoir plus
Résumé

Flavaglines are cyclopenta[b]benzofurans found in plants of the genus Aglaia, several species of which are used in traditional Chinese medicine. These compounds target the initiation factor of translation eIF4A and the scaffold proteins prohibitins-1 and 2 (PHB1/2) to exert various pharmacological activities, including antiviral effects against several types of viruses, including coronaviruses. This review is focused on the antiviral effects of flavaglines and their therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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Karol Kramarz, Anissia Ait Saada, Sarah A E Lambert (2020 Aug 26)

The Analysis of Recombination-Dependent Processing of Blocked Replication Forks by Bidimensional Gel Electrophoresis.

Methods in molecular biology (Clifton, N.J.) : 365-381 : DOI : 10.1007/978-1-0716-0644-5_25 En savoir plus
Résumé

The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing. Applying the bidimensional gel electrophoresis (2DGE) technique to those site-specific RFBs allows the visualization of replication intermediates formed in response to replication fork arrest to investigate the mechanisms ensuring replication fork integrity. Here, we describe the 2DGE technique applied to the site-specific RTS1-RFB in Schizosaccharomyces pombe and explain how this approach allows the detection of arrested forks undergoing nascent strands resection.

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(2020 Aug 26)

Identification and Analysis of Different Types of UFBs

Methods Mol Biol. : DOI : 10.1007/978-1-0716-0644-5_13 En savoir plus
Résumé

Ultrafine anaphase bridges (UFBs) result from a defect in sister chromatid segregation during anaphase. They arise from particular DNA structures, mostly generated at specific loci in the human genome, such as centromeres, common fragile sites, telomeres, or ribosomal DNA. Increases in UFB frequency are a marker of genetic instability, and their detection has become a classic way of detecting such genetic instability over the last decade. Here we describe a protocol to stain different types of UFBs in adherent human cells.

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(2020 Aug 26)

Monitoring Homologous Recombination Activity in Human Cells

.Methods Mol Biol : DOI : 10.1007/978-1-0716-0644-5_9 En savoir plus
Résumé

DNA double-strand breaks (DSBs) are among the most toxic lesions. This type of DNA damage is repaired by two major pathways, homologous recombination (HR), operating only in S/G2 cell-cycle phases and nonhomologous end joining (NHEJ) which is operative throughout the cell cycle. Because HR is a template-directed repair, it is generally less prone to errors and/or translocations than NHEJ.The HR pathway involves several effector proteins and regulators that modulate the efficiency of repair and limit the repair outside S/G2 phase. Some of the genes coding for these proteins are frequently mutated in human diseases such as cancer, and pathogenic mutations or variants identified in patients often alter the HR proficiency of the cells.This chapter describes a cell-based gene-targeting reporter assay in human cells to evaluate the repair of a site-specific DSB by HR . In it, a promoter-less fluorescent protein is encoded in a plasmid flanked by two homology arms directed to a safe-harbour locus in the genome. The expression of the fluorescent protein is driven by the promoter of the endogenous locus enabling to quantify the efficiency of HR by flow cytometry. This approach can be used to determine the requirement of certain proteins, protein domains, or protein modifications for HR . It can also be used to functionally evaluate variants of the genes encoding these proteins such as BRCA1, BRCA2, RAD51C, and PALB2; which may help assess their pathogenicity. Here, we use the homologous recombination mediator BRCA2 to illustrate the assay.

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Sandra Cunha Silveira, Géraldine Buhagiar‑Labarchède, Rosine Onclercq‑Delic, Simon Gemble, Elias Bou Samra, Hamza Mameri, Patricia Duchambon, Christelle Machon, Jérôme Guitton & Mounira Amor‑Guéret (2020 Aug 17)

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

Scientific Reports : 10 : 13907 : DOI : 10.1038/s41598-020-70874-6 En savoir plus
Résumé

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

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(2020 Jun 1)

Preclinical Efficacy of Humanized, non-FcgammaR Binding Anti-CD3 Antibodies in T-cell Acute Lymphoblastic Leukemia

Blood. : DOI : 10.1182/blood.2019003801 En savoir plus
Résumé

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for about 20% ALL cases. Intensive chemotherapy regimens result in >85% cure rates in children and <50% in adults, calling for the search of novel therapeutic strategies. While immune-based therapies have tremendously improved the treatment of B-ALL and other B-cell malignancies, they are not available yet in T-ALL. We report here that humanized, non-FcgR binding monoclonal antibodies to CD3 have anti-leukemic properties in xenograft (PDX) models of CD3+ T-ALL, resulting in prolonged host survival. We also report that these antibodies cooperate with chemotherapy to enhance anti-leukemic effects and host survival. As these antibodies show only minor, manageable side effects in humans, they offer a new therapeutic option for the treatment of T-ALL. Our results also show that the anti-leukemic properties of anti-CD3 mAbs are largely independent Fcg-receptor mediated pathways in T-ALL PDX.

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Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Gaëlle Fontaine, Sarah Lambert, Mounira Amor-Guéret (2020 May 14)

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Open biology : 190259 : DOI : 10.1098/rsob.190259 En savoir plus
Résumé

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

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Åsa Ehlén, Charlotte Martin, Simona Miron, Manon Julien, François-Xavier Theillet, Virginie Ropars, Gaetana Sessa, Romane Beaurepere, Virginie Boucherit, Patricia Duchambon, Ahmed El Marjou, Sophie Zinn-Justin, Aura Carreira (2020 Apr 14)

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1.

Nature communications : 1819 : DOI : 10.1038/s41467-020-15689-9 En savoir plus
Résumé

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

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Samah Matmati, Sarah Lambert, Vincent Géli, Stéphane Coulon (2020 Mar 12)

Telomerase Repairs Collapsed Replication Forks at Telomeres.

Cell reports : 3312-3322.e3 : DOI : S2211-1247(20)30233-3 En savoir plus
Résumé

Telomeres are difficult-to-replicate sites whereby replication itself may threaten telomere integrity. We investigate, in fission yeast, telomere replication dynamics in telomerase-negative cells to unmask problems associated with telomere replication. Two-dimensional gel analysis reveals that replication of telomeres is severely impaired and correlates with an accumulation of replication intermediates that arises from stalled and collapsed forks. In the absence of telomerase, Rad51, Mre11-Rad50-Nbs1 (MRN) complex, and its co-factor CtIP become critical to maintain telomeres, indicating that homologous recombination processes these intermediates to facilitate fork restart. We further show that a catalytically dead mutant of telomerase prevents Ku recruitment to telomeres, suggesting that telomerase and Ku both compete for the binding of telomeric-free DNA ends that are likely to originate from a reversed fork. We infer that Ku removal at collapsed telomeric forks allows telomerase to repair broken telomeres, thereby shielding telomeres from homologous recombination.

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Carsten Janke, Maria M Magiera (2020 Feb 27)

The tubulin code and its role in controlling microtubule properties and functions.

Nature reviews. Molecular cell biology : DOI : 10.1038/s41580-020-0214-3 En savoir plus
Résumé

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the ‘tubulin code’. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.

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Satish Bodakuntla, Anne Schnitzler, Cristopher Villablanca, Christian Gonzalez-Billault, Ivan Bieche, Carsten Janke, Maria M Magiera (2020 Feb 13)

Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons.

Journal of cell science : DOI : jcs241802 En savoir plus
Résumé

Neurons are highly complex cells that heavily rely on intracellular transport to distribute a range of functionally essential cargoes within the cell. Post-translational modifications of tubulin are emerging as mechanisms for regulating microtubule functions, but their impact on neuronal transport is only marginally understood. Here, we have systematically studied the impact of post-translational polyglutamylation on axonal transport. In cultured hippocampal neurons, deletion of a single deglutamylase, CCP1 (also known as AGTPBP1), is sufficient to induce abnormal accumulation of polyglutamylation, i.e. hyperglutamylation. We next investigated how hyperglutamylation affects axonal transport of a range of functionally different neuronal cargoes: mitochondria, lysosomes, LAMP1 endosomes and BDNF vesicles. Strikingly, we found a reduced motility for all these cargoes, suggesting that polyglutamylation could act as a regulator of cargo transport in neurons. This, together with the recent discovery that hyperglutamylation induces neurodegeneration, makes it likely that perturbed neuronal trafficking could be one of the central molecular causes underlying this novel type of degeneration.This article has an associated First Person interview with the first author of the paper.

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Lambert, S. Borde, V. Charbonnier, J. B. Dantzer, F. Espeli, O. Guirouilh-Barbat, J. Llorente, B. Legube, G. Prioleau, M. N. Radicella, P. (2020 Feb 1)

Des mécanismes moléculaires aux applications cliniques. L’essentiel du Colloque Réplication-Réparation-Recombinaison 2019

Bull Cancer : 283-287 : DOI : 10.1016/j.bulcan.2020.01.003 En savoir plus
Iris Tanaka, Alina Chakraborty, Olivier Saulnier, Clara Benoit-Pilven, Sophie Vacher, Dalila Labiod, Eric W F Lam, Ivan Bièche, Olivier Delattre, Frédéric Pouzoulet, Didier Auboeuf, Stéphan Vagner, Martin Dutertre (2020 Jan 17)

ZRANB2 and SYF2-mediated splicing programs converging on ECT2 are involved in breast cancer cell resistance to doxorubicin.

Nucleic acids research : DOI : gkz1213 En savoir plus
Résumé

Besides analyses of specific alternative splicing (AS) variants, little is known about AS regulatory pathways and programs involved in anticancer drug resistance. Doxorubicin is widely used in breast cancer chemotherapy. Here, we identified 1723 AS events and 41 splicing factors regulated in a breast cancer cell model of acquired resistance to doxorubicin. An RNAi screen on splicing factors identified the little studied ZRANB2 and SYF2, whose depletion partially reversed doxorubicin resistance. By RNAi and RNA-seq in resistant cells, we found that the AS programs controlled by ZRANB2 and SYF2 were enriched in resistance-associated AS events, and converged on the ECT2 splice variant including exon 5 (ECT2-Ex5+). Both ZRANB2 and SYF2 were found associated with ECT2 pre-messenger RNA, and ECT2-Ex5+ isoform depletion reduced doxorubicin resistance. Following doxorubicin treatment, resistant cells accumulated in S phase, which partially depended on ZRANB2, SYF2 and the ECT2-Ex5+ isoform. Finally, doxorubicin combination with an oligonucleotide inhibiting ECT2-Ex5 inclusion reduced doxorubicin-resistant tumor growth in mouse xenografts, and high ECT2-Ex5 inclusion levels were associated with bad prognosis in breast cancer treated with chemotherapy. Altogether, our data identify AS programs controlled by ZRANB2 and SYF2 and converging on ECT2, that participate to breast cancer cell resistance to doxorubicin.

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Yanzhang Luo, ShengQi Xiang, Peter Jan Hooikaas, Laura van Bezouwen, A S Jijumon, Carsten Janke, Friedrich Förster, Anna Akhmanova, Marc Baldus (2020 Jan 2)

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

Nature communications : 18 : DOI : 10.1038/s41467-019-13876-x En savoir plus
Résumé

Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [C, N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.

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Année de publication : 2019

Shensi Shen, Sara Faouzi, Amandine Bastide, Sylvain Martineau, Hélène Malka-Mahieu, Yu Fu, Xiaoxiao Sun, Christine Mateus, Emilie Routier, Severine Roy, Laurent Desaubry, Fabrice André, Alexander Eggermont, Alexandre David, Jean-Yves Scoazec, Stéphan Vagner, Caroline Robert (2019 Dec 18)

An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells.

Nature communications : 5713 : DOI : 10.1038/s41467-019-13360-6 En savoir plus
Résumé

Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse. Here we show that a subpopulation of BRAF mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity. Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation. Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells. Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5′-untranslated region of some highly translated mRNAs. Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance.

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