Acute myeloid leukemia (AML) is a highly fatal blood cancer that is characterized by disruption of healthy differentiation of stem cells into functional blood cells in the bone marrow. Most patients with AML consequently die from infections due to the lack of immune cells. For decades, the standard method of remission induction for AML has been chemotherapy using an antineoplastic drug known as AraC. However, even after successful remission induction, aggressive, refractory relapse occurs in the majority of patients within 3 years with dismal survival rates. Here, we sought to develop a genome-wide screening approach to determine the causative genes in AML relapse. In the developed procedure, protein-coding genes of the human genome are screened using a leading-edge technology known as CRISPR (clustered regularly interspaced short palindromic repeats) activation screening. This involves usage of a pooled guide RNA library that upregulates a unique gene for each individual AML cell. By treating these cells with AraC in a mouse xenograft model, the bone marrow will gradually be enriched with cells that carry a guide RNA for a relapse-conducive gene. By harvesting and sequencing all enriched guide RNAs at relapse, the causative genes in AML relapse can be determined. All parameters of the in vivo CRISPR-activation screen have been optimized, and the workflow from preparation to the end of screening has been detailed. Follow-up studies that will validate the results of the screen have also been described. The long-term goal of this developed screen is to elucidate the mechanisms of AML relapse and find ways to clinically target these pathways in conjunction with standard the AraC-based chemotherapy. / Thesis / Master of Science (MSc)

In addition to ATP generation, mitochondria are essential in various cellular processes ranging from biosynthetic pathways, apoptosis, cell cycle progression, and calcium buffering. Studies in living cells have now firmly established that mitochondria exist as a dynamic network sculpted by fission and fusion reactions, rather than separated, individual organelles. Not surprisingly, mutations in genes involved in mitochondrial dynamics and quality control lead to human diseases such as Charcot-Marie-Tooth disease type 2A, Optic atrophy, and autosomal recessive Parkinson disease. I have designed a high-throughput protocol to permit genome-wide screening for novel genes that are required for normal mitochondrial morphology. I have executed a genome-wide RNA interference screen and identified several novel genes required for mitochondrial dynamics in addition to known regulators of mitochondrial dynamics. A detailed high-throughput genome-wide screening protocol is presented. I have shown that TID1, a gene identified from the screen, has a dual-role in maintaining the integrity of mitochondrial DNA and preventing the aggregation of complex I subunits. My analysis of the mitochondrial role of TID1 supports the existence of a TID1- mediated stress response to ATP synthase inhibition. The genome screen also identified the novel gene ROMO1 as essential for normal mitochondrial morphology. I have shown that ROMO1 may have an additional role in maintaining mitochondrial spare respiratory capacity, possibly by affecting cellular substrate availability. Finally, in a collaborative effort, we have shown that homozygous mutations in the mitochondrial fusion gene MFN2 lead to multiple symmetric lipomatosis (MSL) associated with neuropathy. Mechanistically, this mutation reduces MFN2 homocomplex formation. Taken together, these results show the utility of genome-wide screening in identifying genes involved in mitochondrial quality control.

RNA interference (RNAi) is a natural mechanism of cellular defence against exogenous double stranded RNA (dsRNA). The discovery of small dsRNA molecules which can be processed by the RNAi pathway in mammalian cells was one of the key advances in the study of functional genomics. These molecules can be designed to downregulate the expression of specific genes. Collections or libraries of dsRNA molecules targeting an extensive number of genes are now available. Using these libraries, numerous studies have implemented high-throughput screens for the study of molecular effectors of numerous phenotypes. The process of designing an RNAi screen requires the consideration of several critical factors during both the experimental and analysis phases. The experimental screen should aim to reproduce the biological phenomenon studied as closely as possible by choosing an adequate model and screening conditions. Phenotype evaluation and assessment of knockdown effects need careful consideration. The results obtained from large-scale RNAi screens are often complex. An analysis pipeline should be implemented which integrates the biological basis of the phenomenon and facilitates the interpretation of the data. This project designed and implemented an unbiased shRNA screen in two in vitro models relevant to carcinogenesis and diabetes. The first screen implemented used a model of neighbour suppression to study the molecular effectors of the response in tumorigenic cells to growth suppression cues from the surrounding tissue, a cellular interaction relevant in early tumorigenesis. The second screen studied two phenotypes relevant to diabetes: proliferation and resistance to lipotoxicity of β-cells in a reversibly immortalised  cell line. An integrative analysis pipeline was also developed to apply network biology and functional enrichment analysis methods for the interpretation of the data obtained from both screens.

No description available.

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.