Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation, as well as malignant progression of breast cancer cells. Telomerase is an attractive anticancer therapeutic target because its activity is present in over 90% of human cancers, including more than 95% of breast carcinomas, but undetectable in most somatic cells. Traditions chemo- and radio-therapies lack the ability to effectively control and cure breast cancer, in part because residual cells are or become resistant to DNA damaging modalities.While various telomerase inhibition strategies cause cancer cells to undergo apoptosis car senescence, there is often a lag period between administration and biologic effect (Corey, 2002). Our goal in this study was to compare the efficacy of different telomerase inhibition strategies in concert with standard chemotherapeutic agents at triggering senescence and/or apoptosis in cultures of breast cancer cells. We hypothesized that telomerase inhibition strategies will sensitize breast cancer cells to traditional chemotherapies, potentially reducing the lag phase, allowing for more potent anti-tumor effects at lower doses, and therefore ultimately imparting less toxicity to the patient.We blocked telomerase by targeting hTR and hTERT, individually and collectively utilizing synthetic short interfering RNA (siRNA), short hairpin RNA (siRNA), and a dominant negative form of hTERT (DN-hTERT) in MCF-7 breast cancer cells. We analyzed the efficiency of telomerase inhibition for each strategy alone and then treated the cells with two mainstay chemotherapeutic agents, Adriamycin (AdR) and Taxol. The most effective telomerase inhibition strategies were synthetic siRNA and DN-hTERT, individually. After treatment with various concentrations of AdR or Taxol, breast cancer cells with inhibited telomerase grew significantly slower and exhibited widespread senescence or apoptosis within a much shorter time period and at a dose that is insufficient to trigger cytostasis. In addition, we provide evidence that cells in which telomerase was inhibited were more sensitive to anti-cancer agents, whether the drug inhibited topoisomerase II resulting in DNA damage (AdR) or blocked mitosis via protracted microtubule stabilization (Taxol). Collectively, our data indicate that alone, anti-telomerase inhibition strategies differ in their efficacy. However, when used in the adjuvant setting with diverse acting chemotherapeutic agents, there is a potent synergy resulting in chemotherapeutic sensitization characterized in part by widespread senescence and/or apoptosis.

Small molecule mediated chemical intervention of biological processes using nucleic acid targets has proven extremely successful and is continually providing exciting new avenues for the development of anti-cancer agents and molecular probes. Among the alternative DNA confrormations, G-quadruplexes has certainly garnered much recognition due to increase in evidences supporting their involvement in diverse biological process. The grooves of the quadruplexes offer an alternate recognition site for ligand interactions with potentially higher selectivity than the traditional terminal stacking sites. DB832, a bifuryl-phenyl diamidine, was recently reported to selectively recognize human telomeric G-quadruplex, as a stacked species, with significant selectivity over duplex sequences. A series of biophysical studies were conducted to test the groove-binding mode of DB832, along with the selectivity for diverse quadruplex forming sequences. To gain better understanding of quadruplex groove-recognition by DB832, a series of structurally similar heterocyclic diamidines were also evaluated. The unique binding mode of DB832 may allow it to serve as a paradigm for the design of new class of highly selective quadruplex groove-binding molecules. Beyond the alternative secondary structures, it is also becoming increasingly apparent that the structure and dynamics of the canonical Watson–Crick DNA double helix play pivotal roles in diverse biological functions. DB1878, a phenyl-furan-indole diamidine, was shown to recognize a mixed GC/AT motif as a stacked antiparallel dimer, and a detailed structural analysis is reported here. Interestingly, the DNA recognition is completely different from the reported molecules in literature, and represents an entirely new motif for DNA minor groove recognition.

Les extrémités des chromosomes eucaryotes ressemblent à une cassure double brin et, en tant que telles, peuvent conduire à l'activation indésirable de la réponse aux dommages de l'ADN. Les télomères sont une structure ribonucléoprotéique qui coiffe les extrémités des chromosomes et les protège contre l'activation indésirable de la réparation des dommages à l'ADN. Après chaque division cellulaire, on observe un raccourcissement progressif des télomères, ce qui limite leur potentiel prolifératif. Une enzyme spécialisée, la télomérase, reconstitue les télomères pour contrebalancer leur érosion. La télomérase est régulée à la baisse dans la plupart des cellules somatiques. Cependant, l'activité de la télomérase est détectée dans la plupart des cellules souches adultes, bien qu'à de faibles niveaux. Le déficit en télomérase a été associé à un groupe de "troubles de la biologie des télomères" (ou téloméropathies), englobant des maladies de vieillissement prématuré, des syndromes d'insuffisance de la moelle osseuse, des fibroses pulmonaires et des maladies du foie. À l'inverse, dans le cancer, environ 85 % des types de tumeurs sont positifs à la télomérase. Par conséquent, l'inhibition de la télomérase est depuis longtemps considérée comme une cible attrayante pour le traitement du cancer. Dans la présente étude, nous avons cherché à découvrir les facteurs qui affectent la fonction de la télomérase humaine et d'autres protéines associées aux télomères ou à la télomérase. Tout d'abord, nous nous sommes concentrés sur l'identification de nouveaux inhibiteurs de la télomérase à partir de composés naturels. Une nouvelle catéchine a été identifiée dans les extraits végétaux de Burkea africana. Les catéchines sont une classe de molécules que l'on trouve couramment dans le thé vert. La catéchine isolée a inhibé la télomérase humaine recombinante in vitro avec un IC50 de 16,19 μM. Dans un deuxième chapitre, nous avons utilisé un criblage d'édition de bases CRISPR dans une lignée cellulaire humaine pour étudier des mutations cliniquement pertinentes dans 22 gènes importants pour l'homéostasie des télomères. Nous avons identifié des variantes qui affectent négativement l'aptitude cellulaire, y compris certaines variantes précédemment annotées comme variantes de signification incertaine. Nous avons également détecté pour la première fois des variantes hTERT qui confèrent une résistance à la petite molécule BIBR1532, un inhibiteur de la télomérase. Nous avons montré que ces allèles résistants aux médicaments permettent l'immortalisation cellulaire et ont un potentiel tumorigène accru. L'ensemble de ces études souligne l'importance de la télomérase humaine pour le maintien des télomères et la santé cellulaire, contribuant ainsi à une meilleure compréhension du rôle de la télomérase dans le cancer et les troubles de la biologie des télomères. / The extremities of eukaryotic chromosomes resemble a double-stranded break and, as such, can lead to the unwanted activation of the DNA damage response. Telomeres are a ribonucleoprotein structure that caps the ends of the chromosomes and protects them from the unwanted activation of DNA damage recognition and repair processes. After each cellular division, progressive telomere shortening is observed, limiting cellular proliferative potential. A specialized enzyme called telomerase replenishes telomeres to counterbalance telomere erosion. Telomerase is downregulated in most somatic cells. However, telomerase activity is detected in most adult stem cells, although at low levels. Telomerase deficiency has been linked to a group of “Telomere Biology Disorders” (or telomeropathies), encompassing premature aging diseases, bone marrow failure syndromes, pulmonary fibrosis and liver diseases. Conversely, in cancer, around 85% of tumour types are telomerase-positive. Therefore, telomerase inhibition has long been considered an attractive target for cancer therapy. In the present study, we aimed to uncover factors that affect the function of human telomerase and other telomere or telomerase-associated proteins. Firstly, we focused on identifying new telomerase inhibitors from natural compounds. A new catechin was identified in the plant extracts from Burkea africana. Catechins are a class of molecules commonly found in green tea. The isolated catechin inhibited recombinant human telomerase in vitro with an IC50 of 16.19 μM. In the second chapter, we employed a CRISPR base editing screen in a human cell line to investigate clinically-relevant mutations in 22 genes important for telomere homeostasis. We identified variants that negatively affected cell fitness, including some variants previously annotated as variants of uncertain significance. Also, we uncovered hTERT variants that confer resistance to the small molecule BIBR1532, a telomerase inhibitor. We showed that these drug-resistant alleles permit cellular immortalization and exhibit tumorigenic potential at levels comparable to wild-type telomerase. Combined, these studies highlight the importance of human telomerase for telomere maintenance and cell fitness, thereby furthering our understanding of the role of telomerase in cancer and telomere biology disorders.

Le matériel génétique (l’ADN) d’un organisme contient l’information nécessaire à sa survie, sa croissance et sa reproduction. La perte de cette information affecte grandement la santé de l’organisme et cette altération est l’un des facteurs les plus courants dans le vieillissement ou le cancer. Quasiment toutes les cellules d’un organisme contiennent une copie de ce matériel génétique, communément appelé le génome, et font usage de plusieurs mécanismes pour en réparer les sections endommagées ainsi que pour le copier avec précision lors de la division cellulaire. Nous avons cherché à étudier les processus cellulaires qui maintiennent la stabilité génomique en inactivant systématiquement chacun des gènes avec la technique de criblage par CRISPR afin d’en étudier les rôles. Nous avons effectué ces criblages à l’échelle du génome dans des lignées cellulaires humaines en combinaison avec des perturbations chimiques dans le but d’identifier l’effet du traitement chimique ou le rôle de gènes qui exacerbent ou atténuent la perturbation. Nous nous sommes d’abord concentrés sur le resvératrol, une molécule initialement extraite de plantes qui a démontré des propriétés antivieillissement dans certains organismes modèles ainsi que la capacité d’inhiber la prolifération cellulaire. Notre criblage génétique a révélé que le resvératrol inhibait la réplication de l’ADN. En comparant les effets cellulaires du resvératrol à l’hydroxyurée, un agent connu pour causer du stress réplicatif, nous avons montré que ces deux traitements menaient à une diminution similaire de la progression de la fourche de réplication ainsi qu’à une activation de la signalisation en réponse au stress réplicatif. Nous avons également démontré que l’inhibition de la réplication de l’ADN dans les cellules humaines par le resvératrol est l’un des effets principaux de la molécule sur la prolifération cellulaire et ne requiert pas la présence de la déacétylase d’histone Sirtuin-1, protéine qui a été suggérée comme étant la cible principale du resvératrol pour son effet antivieillissement. Nous avons également étudié la perturbation d’un second processus cellulaire, soit le maintien des télomères. Ces séquences spéciales aux extrémités des chromosomes sont indispensables à la protection du génome et leur érosion graduelle est contrebalancée par l’activité enzymatique de la télomérase. Nous avons effectué un crible génétique par CRISPR à l’échelle du génome dans une lignée cellulaire dont nous avons inhibé la télomérase en utilisant BIBR1532, un inhibiteur spécifique de la télomérase. Nous avons découvert une forte interaction génétique entre la télomérase et C16orf72, un gène non-annoté que nous avons nommé TAPR1. Nous avons montré que les cellules déficientes en TAPR1 possèdent des niveaux élevés de la protéine p53, un facteur de transcription central à la réponse cellulaire aux dommages télomériques et aux dommages à l’ADN. Nous suggérons que TAPR1 agit comme un inhibiteur de la stabilité protéique de p53. En somme, ces travaux mettent en évidence la capacité des cribles génétiques CRISPR à approfondir nos connaissances sur le fonctionnement des processus de maintien de la stabilité génomique chez l’humain. / The genetic material (DNA) of an organism contains the necessary information for survival, growth and reproduction. Loss of this information strongly impacts the health of the organism and is the leading factor in aging and cancer. Almost all cells in an organism contain a copy of said genetic material (genome) and employ several mechanisms to repair any damaged section of the genome and to accurately copy it during cell division. We sought to understand the cellular processes by which cells maintain genome stability by systematically inactivating individual genes to uncover their role using pooled CRISPR-Cas9 screening. We employed genome-wide CRISPR screening in human cell lines in combination with specific chemical perturbations to identify gene deletions that enhance or suppress the phenotype of the chemical treatment, thereby shedding light on the effect of the treatment or the role of said enhancer/suppressor genes. We first focused on resveratrol; a small molecule first discovered in plants that has been suggested to extend lifespan in model organisms while also inhibiting cell proliferation ex vivo. Chemical-genetic screening pinpointed a role of resveratrol in inhibition of DNA replication. When we compared the cellular effects of resveratrol to hydroxyurea, a known inducer of replicative stress, we found that both treatments led to slower replication fork progression and activation of signaling in response to replicative stress. Importantly, we showed that the inhibition of DNA replication by resveratrol in human cells is a primary effect on cell proliferation and independent of the histone deacetylase Sirtuin-1, which has been implicated as the primary target in lifespan extension by resveratrol. We then studied the perturbation of a second cellular process, namely telomere maintenance. These specialized sequences at the termini of chromosomes are critical for the protection of chromosome ends and their erosion is counteracted by the enzymatic activity of telomerase. We performed a genome-wide CRISPR screen in cells that were concomitantly treated with a specific telomerase inhibitor, BIBR1532. We uncovered a strong genetic interaction between telomerase and a previously unannotated gene, C16orf72, which we named TAPR1. We found that TAPR1-depleted cells led to elevated p53 levels, a transcription factor central for the cellular response to telomeric and global DNA damage. We propose that TAPR1 is a negative regulator of p53 protein levels by promoting its turnover. Altogether, these studies highlight the power of CRISPR-Cas9 in genetic screening to uncover novel insight into the human genome stability maintenance network.