Academic literature on the topic 'Stabilisation des G4'

G-quadruplexes (G4s) have been identified as a potential alternative chemotherapy target. A series of eight β-amino acid derived naphthalenediimides (NDI) were screened against a series of oncogenic G4 sequences: c-KIT1, h-TELO, and TBA. Three sets of enantiomers were investigated to further our understanding of the effect of point chirality on G4 stabilisation. Enantioselective binding behaviour was observed with both c-KIT1 and h-TELO. Docking studies using GNINA and UV-vis titrations were employed to better understand this selective binding behaviour.

G-quadruplexes (G4) are now extensively recognised as a peculiar non-canonical DNA geometry that plays a prime importance role in processes of biological relevance whose number is increasing continuously. The same is true for the less-studied RNA G4 counterpart. G4s are stable structures; however, their geometrical parameters may be finely tuned not only by the presence of particular sequences of nucleotides but also by the salt content of the medium or by a small molecule that may act as a peculiar topology inducer. As far as the interest in G4s increases and our knowledge of these species deepens, researchers do not only verify the G4s binding by small molecules and the subsequent G4 stabilisation. The most innovative studies now aim to elucidate the mechanistic details of the interaction and the ability of a target species (drug) to bind only to a peculiar G4 geometry. In this focused review, we survey the advances in the studies of the binding of small molecules of medical interest to G4s, with particular attention to the ability of these species to bind differently (intercalation, lateral binding or sitting atop) to different G4 topologies (parallel, anti-parallel or hybrid structures). Some species, given the very high affinity with some peculiar G4 topology, can first bind to a less favourable geometry and then induce its conversion. This aspect is also considered.

Parvovirus B19 (B19V), an ssDNA virus in the family Parvoviridae, is a human pathogenic virus, responsible for a wide range of clinical manifestations, still in need of effective and specific antivirals. DNA structures, including G-quadruplex (G4), have been recognised as relevant functional features in viral genomes, and small-molecule ligands binding to these structures are promising antiviral compounds. Bioinformatic tools predict the presence of potential G4 forming sequences (PQSs) in the genome of B19V, raising interest as targets for antiviral strategies. Predictions locate PQSs in the genomic terminal regions, in proximity to replicative origins. The actual propensity of these PQSs to form G4 structures was investigated by circular dichroism spectroscopic analysis on synthetic oligonucleotides of corresponding sequences. No signature of G4 structures was detected, and the interaction with the G4 ligand BRACO-19 (N,N′-(9-{[4-(dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1-ylpropanamide) did not appear consistent with the stabilisation of G4 structures. Any potential role of PQSs in the viral lifecycle was then assessed in an in vitro infection model system, by evaluating any variation in replication or expression of B19V in the presence of the G4 ligands BRACO-19 and pyridostatin. Neither showed a significant inhibitory activity on B19V replication or expression. Experimental challenge did not support bioinformatic predictions. The terminal regions of B19V are characterised by relevant sequence and symmetry constraints, which are functional to viral replication. Our experiments suggest that these impose a stringent requirement prevailing over the propensity of forming actual G4 structures.

The stabilisation of G-quadruplexes (G4s) by small-molecule compounds is an effective approach for causing cell growth arrest, followed by cell death. Some of these compounds are currently being developed for the treatment of human cancers. We have previously developed a substituted naphthalene diimide G4-binding molecule (CM03) with selective potency for pancreatic cancer cells, including gemcitabine-resistant cells. We report here that CM03 and the histone deacetylase (HDAC) inhibitor SAHA (suberanilohydroxamic acid) have synergistic effects at concentrations close to and below their individual GI50 values, in both gemcitabine-sensitive and resistant pancreatic cancer cell lines. Immunoblot analysis showed elevated levels of γ-H2AX and cleaved PARP proteins upon drug combination treatment, indicating increased levels of DNA damage (double-strand break events: DSBs) and apoptosis induction, respectively. We propose that the mechanism of synergy involves SAHA relaxing condensed chromatin, resulting in higher levels of G4 formation. In turn, CM03 can stabilise a greater number of G4s, leading to the downregulation of more G4-containing genes as well as a higher incidence of DSBs due to torsional strain on DNA and chromatin structure.

Since the occurrence of tumours is closely associated with the telomerase function and oncogene expression, the structure of such enzymes and genes are being recognized as targets for new anticancer drugs. The efficacy of several ligands in telomerase inhibition and in the regulation of genes expression, by an effective stabilisation of G-quadruplexes (G4) DNA structures, is being considered as a promising strategy in cancer therapies. When evaluating the potential of a ligand for telomerase inhibition, the selectivity towards quadruplex versus duplex DNA is a fundamental attribute due to the large amount of double-stranded DNA in the cellular nucleus. This study reports the evaluated efficacy of three tetracationic opp-dibenzoporphyrins, a free base, and the corresponding zinc(II) and nickel(II) complexes, to stabilise G4 structures, namely the telomeric DNA sequence (AG3(T2AG3)3). In order to evaluate the selectivity of these ligands towards G4 structures, their interaction towards DNA calf thymus, as a double-strand DNA sequence, were also studied. The data obtained by using different spectroscopic techniques, such as ultraviolet-visible, fluorescence, and circular dichroism, suggested good affinity of the free-base porphyrin and of its zinc(II) complex for the considered DNA structures, both showing a pattern of selectivity for the telomeric G4 structure. A pattern of aggregation in aqueous solution was detected for both Zn(II) and Ni(II) metallo dibenzoporphyrins and the ability of DNA sequences to induce ligand disaggregation was observed.

4041 Background: Activating MAPK pathway mutations (predominantly RAS) occur with a high incidence in metastatic pancreatic adenocarcinoma (mPaCa). Pimasertib is a MEK1/2 inhibitor with potent activity in cell lines and xenografts with an activated MAPK pathway. This two-part trial in patients (pts) with mPaCa comprises a dose-escalation safety run-in and a randomized phase II part (EudraCT 2009-011992-61). We defined the maximum tolerated dose (MTD), safety, pharmacokinetics (PK) and antitumor activity of two pimasertib dosing schedules (S), and the recommended phase II dose (RP2D). Methods: Dose-escalation (3+3 design) in two dosing S of oral pimasertib: once-daily (qd) - 5 days on, 2 days off (S1); and twice-daily (bid) - continuous (S2) combined with the standard dose of gemcitabine (gem). Results: 53 pts (median age 61 years and ECOG performance status 0-1) have been treated at six dose levels in S1 (15 to 120 mg qd) and at 60 and 75 mg bid in S2. MTDs were defined as 120 mg qd and 75 mg bid. Two pts had a dose-limiting toxicity (DLT) in the DLT observation period: a grade (G) 3 confusion with ataxia and disorientation at 60 mg bid and a G4 suicidal ideation at 75 mg bid. G3-4 adverse events (AEs) in >5% of pts were: neutropenia (32%), thrombocytopenia (25%), asthenia (19%), dyspnea (9%), transaminitis (9%), anemia (8%), and diarrhea, pulmonary embolism, pulmonary sepsis (6% each). Most common AEs were asthenia (70%), ocular AEs (68%), skin rash (62%), nausea (58%), diarrhea (58%), peripheral edema (51%), thrombocytopenia (49%), vomiting (45%), mucositis (43%), neutropenia (38%), decreased appetite (36%) and anemia (34%). The main ocular AE was serous retinal detachment (58%); manageable retinal vein occlusion occurred in five pts. PK data were comparable to pimasertib monotherapy and published gem data. Partial responses were noted in 10 pts and stabilisation ≥3 months in 13 pts. Hot spot mutations in genes activating the MAPK and PI3K/AKT pathway and correlation with clinical outcome are being investigated. Conclusions: Pimasertib MTDs were reached. The RP2D was defined as 60 mg bid. PK was dose proportional and associated with target inhibition. Sustained responses were seen in both dosing schedules. Clinical trial information: 2009-011992-61.

Abstract Abstract 4945 Introduction Current goals in MM-treatment are to achieve prolonged remission- and treatment-free-intervals and to transform the disease into an indolent course. Effective anti-MM-substances are IMIDs, such as thalidomide and lenalidomide, the latter showing a different mode of action and favorable side-effect profile. Lenalidomide-induced side effects may, however, increase with renal impairment (RI) and dose adjustments are recommended, thus sensitive RI-monitoring is desirable. We assessed renal function (RF) via estimated glomerular filtration rate (eGFR by MDRD) and correlated this with lenalidomide-response, since we have shown that eGFR-monitoring is a sensitive method to detect RI in various hematological and solid tumor- (Kleber M. et al., Ann Oncol 2007) and explicitly valuable in MM-pts (Kleber M. et al., EJH 2009). Methods Thirty-two consecutive MM pts received lenalidomide in G1-4 treatment groups at our center between 6/2006-7/2009 (G1=lenalidomide 25mg/dex 40mg [n=12]; G2: lenalidomide 25mg/low-dose-dex-[n=10]; G3: lenalidomide 10mg [n=7]; G4: lenalidomide plus chemotherapy [n=3]). Serum creatinine and eGFR were determined before lenalidomide and after 1, 3 and 6 months. RI was assessed by NKDOQI- and MM-response according to EBMT-criteria. Results The median pt age was 67 years (range; 44-78), with most having stage II/III disease by Durie&Salmon (97%; ISS II/III: 75%). IgG-myeloma and normal cytogenetics were present in 84% and 56%, respectively. Pretreatment was considerable with ≥2 previous therapy lines in 71% and autologous or allogeneic stem cell transplantations performed in 62% and 12%, respectively. Median β2-MG- and hemoglobin-levels were 3.4mg/dl and 11.3mg/dl, respectively. Before lenalidomide-initiation, RF appeared normal with median creatinine levels of 1.0mg/dl (range; 0.6-2.7), nevertheless, mild RI was readily detectable via eGFR (81ml/min/1.73m2, range; 27-119). Of note, mild (eGFR<90) and moderate RI (eGFR<60) before lenalidomide-initiation was prominent in 65% and 31% of pts, respectively. In pts achieving a PR due to lenalidomide treatment, median creatinine-values before and during treatment (1, 3 and 6 months) decreased from 1.0mg/dl to 0.9, 0.9 and 0.8mg/dl, which was even more prominently detectable via eGFR with 71 at baseline, increasing to 86, 85 and 97ml/min/1.73m2, respectively. Pts with SD showed creatinine-values before and during treatment (1, 3 and 6 months) of 0.9 at baseline, increasing to 1.1, 1.0 and 1.1mg/dl, whereas eGFR changed from 86 to 69, 77 and 70ml/min/1.73m2, respectively (Fig. 1). With PD, the creatinine increased from 0.9 to 1.3mg/dl, and eGFR values substantially deteriorated from 87 to 61ml/min/1.73m2; this demonstrating that with lenalidomide response, RI recovered and response was readily detected via eGFR-assessment. The median lenalidomide treatment duration lasted 36 weeks (range; 4-96), inducing an ORR (CR/PR) in 31% (n=10), clinical benefit rate (CBR=CR+PR+MR) in 37% (n=12) and stunning disease stabilisation rate (CBR+SD) in 97% (n=31 pts), this comparing favorably with previous reports, albeit our pt cohort was older, more pretreated and bearing substantial comorbidities than in both trials leading to lenalidomide/dex-FDA-approval. Lenalidomide was well tolerated with WHO-CTC-grade (G) ≥2 side effects in 8 pts (25%): 3 showed neutropenia (G2+3), 2 pts skin rash/exanthema (G2), and disorientation (G2), GvHD-exacerbation (G3) and atrial fibrilation (G3) in each one pt. Conclusions We highlight the importance to detect RI by means of eGFR-assessment, which allows to identify mild and moderate RI more prominently and reliably than via creatinine determination. Our results underline that lenalidomide in elderly MM pts is feasible and well tolerated. Renal improvement by eGFR analysis was associated with lenalidomide-response which is currently being assessed in even more detail. Disclosures Kleber: Celgene: Research Funding. Haas:Celgene: Research Funding. Engelhardt:Celgene: Research Funding.

G-quadruplexes (G4) are alternative nucleic acid structures involved in transcription, translation and replication. Aberrant G4 formation and stabilisation is linked to genome instability and cancer. G4 ligand treatment disrupts key biological processes leading to cell death. To discover genes and pathways involved with G4s and gain mechanistic insights into G4 biology, we present the first unbiased genome-wide study to systematically identify human genes that promote cell death when silenced by shRNA in the presence of G4-stabilising small molecules. Many novel genetic vulnerabilities were revealed opening up new therapeutic possibilities in cancer, which we exemplified by an orthogonal pharmacological inhibition approach that phenocopies gene silencing. We find that targeting the WEE1 cell cycle kinase or USP1 deubiquitinase in combination with G4 ligand treatment enhances cell killing. We also identify new genes and pathways regulating or interacting with G4s and demonstrate that the DDX42 DEAD-box helicase is a newly discovered G4-binding protein.

Abstract G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in guanine (G)-rich genomic regions. X-linked dystonia parkinsonism (XDP) is an inherited neurodegenerative disease in which a SINE–VNTR–Alu (SVA) retrotransposon, characterised by amplification of a G-rich repeat, is inserted into the coding sequence of TAF1, a key partner of RNA polymerase II. XDP SVA alters TAF1 expression, but the cause of this outcome in XDP remains unknown. To assess whether G4s form in XDP SVA and affect TAF1 expression, we first characterised bioinformatically predicted XDP SVA G4s in vitro. We next showed that highly stable G4s can form and stop polymerase amplification at the SVA region from patient-derived fibroblasts and neural progenitor cells. Using chromatin immunoprecipitazion (ChIP) with an anti-G4 antibody coupled to sequencing or quantitative PCR, we showed that XDP SVA G4s are folded even when embedded in a chromatin context in patient-derived cells. Using the G4 ligands BRACO-19 and quarfloxin and total RNA-sequencing analysis, we showed that stabilisation of the XDP SVA G4s reduces TAF1 transcripts downstream and around the SVA, and increases upstream transcripts, while destabilisation using the G4 unfolder PhpC increases TAF1 transcripts. Our data indicate that G4 formation in the XDP SVA is a major cause of aberrant TAF1 expression, opening the way for the development of strategies to unfold G4s and potentially target the disease.

Les quadruplexes de guanines (G4) sont des structures polymorphiques adoptées in vitro par les séquences d’ADN et d’ARN riches en guanines. L’utilisation d’anticorps et de ligands spécifiques des structures G4 a permis leur détection au niveau cellulaire. Des études computationnelles ont prédit des séquences possédant une signature G4 au niveau de régions génomiques capitales comme les télomères ou les promoteurs de certains oncogènes. De plus, de nombreuses protéines impliquées dans des processus cellulaires comme la réplication, la transcription ou encore la réparation, peuvent interagir directement avec des G4, en facilitant leur formation ou au contraire leur dénaturation. C’est notamment le cas d’hélicases impliquées dans des pathologies humaines, comme BLM, WRN, FANC J ou PIF1. Ce sont des enzymes capables de dénaturer des G4 et dont l'inactivation induit une instabilité génomique, en particulier au niveau de régions susceptibles de former un G4. Dans ce travail, nous présentons la mise au point d’un test de criblage à moyen débit pour le suivi des interactions G4/hélicases en temps réel. Ce test nous a permis de définir les conditions favorisant ou inhibant l’interaction d’une hélicase vis-à-vis de son substrat G4. Nous avons démontré que ces conditions pouvaient différer d’une hélicase à une autre, notamment les conditions salines optimales nécessaires aux activités hélicases de ScPif1 et de RHAU. Nous avons également prouvé, à travers ce test, que l’utilisation de ligands capables de stabiliser les G4 n’induisait pas forcément d’inhibition de l’activité hélicase de ScPif1. Enfin, nous avons également pu définir la directionnalité de la protéine RPA, ce qui fait de notre test une technique prometteuse pour la caractérisation de nouvelles protéines pouvant dérouler des structures G4
G-quadruplexes are highly polymorphic non-canonical nucleic acid structures adopted by both DNA and RNA guanine-rich sequences in vitro. They have been detected at the cellular level using structure specific antibodies and small molecule ligands. Computational studies demonstrated that G4-prone sequences are located in key genomic regions such as telomeres and oncogene promoters. Numerous studies showed that G4 sequences can interact with proteins involved in cellular processes, including replication, transcription or reparation. Those interactions include binding, G4 folding promotion or in contrary unwinding. Indeed, WRN, BLM, FANC J or Pif1 are helicases associated with human-diseases. They can unwind G4 forming sequences; mutation of these helicases lead to genomic instability of G4-prone motifs when mutated. Here, we present a medium-throughput technique to monitor G4-helicase interactions in real time. We were able to determine both favourable and deleterious conditions for G4 unwinding by a given helicase. We show that these conditions differ from one helicase to another as exemplified with the optimal salt conditions required for both ScPif1 and RHAU activities. We also reveal that the G4 ligands that stabilize G4 structures do not necessarily induce an inhibition of their unwinding by ScPif1 helicase. Finally, we also prove that our assay is adapted to clear up RPA directionality, making it an attractive technique to screen for new proteins able to unwind G4 structures

Les G-quadruplexes (ou G4) sont des structures non canoniques des acides nucléiques formées à partir de séquences riches en guanines. Les G4 sont des structures stables, présentes sur l'ensemble du génome et qui peuvent adopter différentes conformations. La formation des G4 peut réguler, de façon positive ou négative, différents processus cellulaires tels que la transcription, la réplication, les transactions des ARN et les mécanismes mitochondriaux. L'ensemble de ces processus nécessite le recrutement de protéines capables de moduler la formation de ces structures. Certaines protéines, telles que les hélicases BLM, WRN ou DHX36, sont capables de dérouler les G4 alors que d'autres, comme la nucléoline (NCL), se lient aux G4 et les stabilisent. Enfin, des molécules capables de stabiliser les G4 appelées ligands de G4, peuvent impacter divers processus dans lesquels sont impliqués les G4 ; en particulier, ils peuvent entrainer la répression de l'expression d'oncogènes et mener à de l'instabilité génomique. Ainsi, les ligands de G4 sont considérés comme de potentiels agents anti-cancéreux. Mes travaux de thèses s'articulent autour de plusieurs problématiques concernant les G4 : 1/ l'amélioration des ligands de G4 et leur caractérisation ; 2/ le décryptage des mécanismes induisant de l'instabilité génomique suite à la stabilisation des G4 par des ligands ; 3/ l'identification des protéines capables de se lier aux G4 (ou GBP pour " G4 Binding Proteins "). Par des expériences biochimiques et biophysiques, j'ai participé à la caractérisation de ligands dérivés de porphyrine. Dans le cas du ligand AuMA, j'ai montré une augmentation à la fois de la capacité de stabilisation des G4 et de la spécificité envers les G4, par rapport à d'autres molécules dérivées de porphyrine. Cette molécule représente donc un meilleur potentiel thérapeutique que le TMPyP4, ligand largement étudié, dont elle est dérivée. J'ai également étudié l'instabilité génomique due à la stabilisation des G4 grâce à l'utilisation du ligand pyridostatine et du ligand CX5461, actuellement en phase II d'un essai clinique. Ces ligands induisent des cassures double brin de l'ADN (ou CDB) dépendantes de la transcription par l'ARN polymérase II et partiellement dues à la pause transcriptionnelle. Les CDB sont initiées par l'activité des Topoisomérases II, enzymes impliquées dans la résolution des stress topologiques de l'ADN dus à la transcription et à la réplication. Ces résultats montrent le rôle important de la transcription dans l'induction de l'instabilité génomique et ouvrent de nouvelles pistes thérapeutiques, dans le traitement de cancers dans lesquels ces protéines sont surexprimées ou par la combinaison avec d'autres chimiothérapies telles que l'étoposide afin d'en augmenter le potentiel cytotoxique. J'ai étudié les protéines se liant aux G4 grâce à des structures contraintes, bloquées dans une conformation particulière, en mettant au point un protocole de détection des GBP par des expériences de "Pull-Down" suivie d'une analyse par spectrométrie de masse. Ces résultats, validés par la liaison aux G4 de protéines déjà identifiées et caractérisées telles que WRN, DHX36 ou encore CNBP, ont permis l'identification de 425 GBP. Ainsi, j'ai mis en évidence de nouvelles GBP impliquées dans divers processus cellulaires tels que la réplication, la réparation de l'ADN, la transcription et le métabolisme des ARN. De façon annexe, l'étude de la protéine CNBP dans un modèle animal a permis de montrer que la régulation des G4 in vivo impacte la transcription et le développement embryonnaire, renforçant le rôle des G4 dans des organismes vivants. Mes travaux contribuent à étendre les connaissances sur les G4 et leurs ligands, particulièrement celles portant sur les mécanismes d'action des G4 pendant la transcription, et ouvrent de nouvelles perspectives thérapeutiques
G-quadruplexes (or G4) are non-canonical structures of nucleic acid formed from guanine-rich sequences. G4 are stable structures, present throughout the genome and could be folded into different conformations. G4 formation can regulate, positively or negatively, different cellular processes such as transcription, replication, RNA transactions and mitochondrial mechanisms. All these processes require the recruitment of proteins able to modulate the formation of these structures. Indeed, some proteins, such as BLM, WRN or DHX36 helicases, are able to unwind G4 while others, like nucleolin (NCL), bind to and stabilize G4. Finally, G4 ligands, small molecules stabilizing G4, can impact various processes in which G4 are involved; in particular, they can cause repression of oncogene expression and lead to genomic instability. Thus, G4 ligands are considered to be potential anti-cancer agents. My thesis work focuses on several issues concerning G4: 1/ the improvement of G4 ligands and their characterization; 2/ the deciphering of the mechanisms inducing genomic instability following G4 stabilization by ligands; 3/ the identification of proteins able to bind to G4 (or GBPs for "G4 Binding Proteins"). Through biochemical and biophysical experiments, I have participated in the characterization of porphyrin-derived ligands. In the case of the AuMA ligand, I showed an increase in both G4 stabilization capacity and G4 specificity, compared to other porphyrin-derived molecules. This molecule therefore represents a better therapeutic potential than TMPyP4, a widely characterized ligand from which it is derived. I have also studied the genomic instability due to G4 stabilization using the pyridostatin ligand and the CX5461 ligand, currently in Phase II of a clinical trial. These ligands induce DNA double-strand breaks (or DSBs) dependent on transcription by RNA polymerase II and partly due to the transcriptional pausing. DSBs are initiated by the activity of Topoisomerases II, enzymes involved in the resolution of DNA topological stresses due to transcription and replication. These results show the significant role of transcription in the induction of genomic instability and open up new therapeutic approaches in the treatment of cancers in which these proteins are overexpressed or by combining them with other chemotherapies such as etoposide to increase their cytotoxic potential. I have studied G4-binding proteins using constrained structures, blocked in a particular conformation, by developing a protocol for the detection of GBPs through Pull-Down experiments followed by mass spectrometry analysis. These results, validated by the binding to G4 of proteins already identified and characterized such as WRN, DHX36 or CNBP, allow the identification of 425 GBP. Thus, I have highlighted new GBPs involved in various cellular processes such as replication, DNA repair, transcription and RNA metabolism. Aside, the study of CNBP protein in a zebrafish model has shown that the regulation of G4 in vivo affects transcription and embryonic development, reinforcing the role of G4 in whole living organisms. My work contributes to extend the knowledge of G4 and their ligands, particularly the mechanisms of action of G4 during transcription, and is opening up new therapeutic perspectives

Les G-quadruplex (G4), structures secondaires pouvant être adoptées par les acides nucléiques, jouent des rôles biologiques dans de nombreux processus cellulaires : maintenance des télomères, réplication cellulaire et virale, réarrangements génomiques, réponse au dommages de l’ADN, régulation transcriptionnelle. Dans le génome humain, il existe une forte présence de séquences potentiellement capables de former des G4 et, en fonction des algorithmes de recherche utilisés pour les identifier in silico, le nombre de motifs est variable et peut atteindre plusieurs centaines de milliers. Récemment, le développement de ligands qui se lient spécifiquement aux G4 et sont capables de stabiliser ces structures, permettrait de moduler leur formation in vivo et donc d’étudier les processus biologiques associés et de développer des agents anti-cancéreux visant des proto-oncogènes.Des travaux menés dans notre unité, basés sur un modèle levure (insertion de la séquence du minisatellite humain CEB25 dans le génome de Saccharomyces cerevisiae), ont montré que les divers motifs G4 ne sont pas tous capables d’induire de l’instabilité génétique et qu’ils n’ont pas le même comportement vis-à-vis des ligands. En particulier, les G4 composés de boucles courtes - dits G4-L1, du type G3N1G3N1G3N1G3, sont beaucoup plus stables in vitro (Tm > 70°C) et corrélativement sont plus instables in vivo. Ces observations mènent à la question de comment ces motifs ‘à risque’ se forment in vivo, sont maintenus dans les génomes et comment ils évoluent. Pour aborder ces questions, nous avons mis au point et employé des approches expérimentales et bio-informatiques pour : (1) localiser, annoter et comparer les motifs G4-L1 dans le génome humain, en utilisant le génome de référence hg38 et les données du projet 1000 Génomes pour évaluer leur polymorphisme, ainsi qu’examiner leur maintien dans plus de 500 espèces ; (2) caractériser le potentiel mutagène des G-quadruplex par une approche de capture-NGS (séquençage à très haute profondeur), en utilisant diverses lignées cellulaires humaines traitées par des ligands de G4 (notamment, les ligands de la famille de PhenDC produits à l’Institut Curie) ; et (3) améliorer notre compréhension des rôles des G4 stables dans la transcription, en associant l'expression génique à la fréquence de motifs G4 canoniques présents autour des TSS, par des analyses combinées de génomique/traitements avec des ligands dans des cellules de fibrosarcome
Nucleic acid G-quadruplex (G4) secondary structures play important biological roles in multiple cellular processes (telomere maintenance, cellular and viral replication, genome rearrangements, DNA damage response, epigenetic, transcriptional regulation). G4 potential motifs are scattered throughout the genome and, depending on the algorithms used to identify them in silico, their number varies and may reach several hundreds of thousands. Recently, the development of small molecules able to stabilise these structures opened the possibility of probing and/or interfering with their formation, allowing to examine the associated biological processes and developing anti-cancer agents. Work in our research unit, using natural and site-directed mutated human minisatellite sequences in yeast treated with different G4 ligands, showed that not all G4 sequences display the same potential to induce G4-dependent genome instability during replication. Namely, G4 structures carrying very short interconnecting loops between the G-runs are more stable in vitro and correlatively, are more prone to trigger genome instability in vivo. At the extreme, the G4s with the consensus G3N1G3N1G3N1G3 motif (where N is any nucleotide) – herein called G4-L1 – carry the higher potential to fold and trigger instability. This raises the unresolved questions of how these ‘at risk’ sequences are formed in vivo, maintained in genomes and how they evolve. To address these issues, we have used up–to-date bioinformatics, analytical and biological approaches to: (1) map, annotate and compare G4-L1 motifs in the human genome, using the hg38 reference and the 1000 Genomes project variation information to assess common polymorphism, as well as examine their maintenance across over 500 species; (2) characterize, using various human cell lines and Next Generation Sequencing (NGS) approaches, the potential of these sequences to mutate or induce perturbations upon treatments with G4 ligands; and (3) to improve our understanding of the roles of stable G4s in transcription, we have associated gene expression with the frequency of canonical G4 motifs near gene by combined G4 ligand/genomic analyses in fibrosarcoma cells

Les cancers du sein triple négatifs et du pancréas sont associés à de faible taux de survie, dû à leur forte résistance aux traitements conventionnels, constituant un réel problème de santé publique et rendant le développement de nouvelles thérapies ciblées crucial. Au niveau des séquences télomériques et des promoteurs d'oncogènes comme cMYC, cKIT et BCL2, les séquences riches en guanines peuvent former des structures secondaires non conventionnelles, appelées G-quadruplexes. Ces structures jouent un rôle important dans la régulation de l'expression génique, constituant ainsi de prometteuses cibles thérapeutiques pour la lutte contre le cancer.Ici, nous avons choisi une approche pluridisciplinaire, alliant synthèse chimique, chimie théorique, et biologie cellulaire et moléculaire, afin d'identifier de nouveaux composés stabilisant ces structures, dans le but de contrôler la prolifération des cellules cancéreuses. Notre laboratoire a précédemment montré l'intérêt des complexes métalliques symétriques et planaires dans la stabilisation spécifique des G-quadruplexes. Ainsi, nous avons synthétisé 12 nouveaux complexes à base des métaux de transition Zn2+, Ni2+, Cu2+, Pd2+ et Pt2+. Leur capacité à sélectivement stabiliser les G-quadruplexes, en comparaison avec l'ADN double brin, a été démontré et des simulations de dynamique moléculaire révèlent un mode d'interaction peu conventionnel, impliquant la boucle du G-quadruplex.Nos composés induisent la formation de G-quadruplexes au sein des lignées cellulaires cancéreuses, entrainant une régulation à la baisse de nombreux oncogènes comme kRAS, RET et cMYC. Cette répression entraine une réduction de la prolifération et la viabilité des cellules cancéreuses, mais n'affecte que peu les cellules saines.Alors que certains composés induisent la mort des cellules cancéreuses par apoptose sans affecter les cellules saines, et inhibent drastiquement l'expression des oncogènes hRAS et cMYC, d'autres complexes causent des dommages à l'ADN dans les cellules néoplasiques pancréatiques T3M4. Aussi, les composés de Zn2+ favorisent l'expression de VEGF-A, en stimulant sa transcription. L'étude de l'impact d'une stabilisation des G-quadruplexes sur la polarisation des macrophages a montré que les composés de nickel promeuvent la polarisation des macrophages naïfs vers un phénotype anticancéreux M1, tout en inhibant l'acquisition de marqueurs pro-tumoraux de type M2.L'ensemble de nos résultats démontre le fort potentiel de nos complexes métalliques en tant que stabilisateurs de G-quadruplexes, présentant de prometteuses propriétés anticancéreuses, notamment en modulant le microenvironnement tumoral. Ces résultats ouvrent la possibilité à de nombreuses perspectives d'investigation, suggérant de nouvelles pistes thérapeutiques en cancérologie
Triple-negative breast cancer and pancreatic adenocarcinoma are associated to very low survival-rates due to their high resistance to conventional treatments, posing significant public healthiness issue. The development of new targeted therapeutic options is then crucial. G-rich sequences in nucleic acids can form non-conventional secondary structures, known as G-quadruplexes, identified in telomeric sequences and in the promoters of potent oncogenes, such as cMYC, cKIT, and BCL2. These structures play a critical role in regulating gene expression, making them as promising therapeutic targets in cancer treatment.In this study, we employed a transdisciplinary approach, integrating chemical synthesis, molecular dynamic simulations, and cellular and molecular biology, to identify novel G-quadruplex binders and stabilizers aimed at controlling cancer progression. Previous work in our laboratory demonstrated that symmetric planar metal complexes could specifically bind these structures. In that sense, we synthesized 12 new transition metal complexes of Zn2+, Ni2+, Cu2+, Pd2+ and Pt2+, from the Salphen scaffold. Their ability to selectively bind and stabilize G-quadruplexes over double-stranded DNA were confirmed. Molecular dynamic simulations revealed an unconventional binding mode involving interaction with the G-quadruplex loop.Immunofluorescence assays confirmed that the compounds enhance G-quadruplex formation, in cancer cell lines, leading to the early downregulation of several G-quadruplex-driven oncogenes, such as kRAS, RET, and cMYC. This downregulation reduced cancer cell proliferation and viability, with less effect on non-cancerous cells.Some complexes induced apoptosis in cancer cells without affecting the non-neoplastic cells, after decreased hRAS and cMYC transcript levels, while other compounds caused DNA damage in pancreatic cancer cells T3M4. Notably, Zn2+ compounds increased VEGF-A expression, enhancing its transcription. We also investigated the effects of G-quadruplex stabilization on macrophages polarization, showing that nickel compounds promoted the polarization of M0 macrophages towards the anticancer M1 phenotype, while inhibiting the acquisition of pro-tumoral M2 markers.Overall, our novel metal complexes demonstrate significant potential in stabilizing G-quadruplex and exhibit promising anticancer properties, including modulation of the tumor microenvironment. These preliminary results suggest avenues for further research, with potential implications for advancing strategies in cancer therapy