By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer. / October 2016

Os Papilomavírus Humanos (HPV) são vírus de DNA, não envelopados que infectam as células epiteliais. A infecção persistente por alguns tipos de HPV é o principal fator de risco para o desenvolvimento do câncer cervical. A maquinaria de reparo de DNA desempenha um papel essencial em várias fases do ciclo de vida do HPV e é crucial para a sobrevivência de células tumorais. Durante a transformação maligna, as oncoproteínas E6 e E7 de HPV são capazes de induzir alterações cromossômicas e numéricas, além de modular a resposta de danos ao DNA. Estas observações sugerem que a maquinaria celular de reparo de dano ao DNA podem desempenhar um papel duplo na biologia do HPV e na sua patogênese. No presente estudo, procurou-se investigar o papel das proteínas de reparo de DNA na biologia das células derivadas de câncer cervical. A fim de alcançar este objetivo, a expressão de 189 genes foi silenciada em células HeLa (HPV 18) e em células SiHa (HPV16), bem como em queratinócitos humanos primários (QHP), utilizando vetores lentivirais que expressam shRNAs específicos. O efeito do silenciamento gênico foi determinado por ensaios de viabilidade celular, análise de proliferação celular, ensaio clonogênico e de formação de colônias em soft ágar. Observamos que o silenciamento dos genes ATM, BRCA1, CHEK2 e HMGB1 reduziu a taxa de crescimento celular, o potencial de crescimento em colônia e a capacidade de crescimento independente de ancoragem das linhagens celulares derivadas de câncer cervical transformadas por HPV, sem afetar QHP. O tratamento das linhagens celulares com fármacos capazes de inibir a atividade das proteínas ATM e CHEK2 revelou uma maior sensibilidade das células tumorais à inibição destas proteínas quando comparadas a QHP. Além disso, mostramos que QHP que expressavam E6E7 ou somente E6 de HPV16 foram mais sensíveis a estes inibidores, quando comparados ao controle QHP ou QHP expressando apenas E7. Além disso, QHP que expressavam mutantes de E6 de HPV16, defectivos para a degradação de p53, foram menos sensíveis do que QHP, que expressavam HPV16 E6 selvagem. Desta forma, estes resultados indicam que estes genes são necessários para a sobrevivência de células transformadas por HPV. Além disso, os nossos resultados sugerem que este efeito está relacionado com a expressão oncoproteína de HPV16 E6 e a sua capacidade para degradar p53. / Human Papillomaviruses (HPV) are non-enveloped DNA viruses that infect epithelial cells. Persistent infection with some HPV types is the main risk factor for the development of cervical cancer. DNA repair machinery plays an essential role in several stages of the HPV life cycle and is crucial for tumor cells survival. During malignant transformation, HPV E6 and E7 oncoproteins induce structural and numerical chromosome alterations and modulate DNA damage response. These observations suggest that cellular DNA repair machinery may play a dual role in both HPV biology and pathogenesis. In the present study, we sought to investigate the role of DNA repair proteins in cervical cancer derived cells biology. In order to achieve this goal, the expression of 189 genes was silenced in HeLa (HPV18) and SiHa (HPV16) cells as well as in primary human keratinocytes (PHK) using lentiviral vectors expressing specific shRNA. The effect of gene silencing was determined by cell viability assay, cell growth analysis, clonogenic and soft agar colony formation test. We observed that ATM, BRCA1, CHEK2 and HMGB1 down-regulation decreased growth rate, clonogenic potential and cellular anchorage-independent growth of HPV-transformed cervical cancer-derived cell lines with no effect in normal keratinocytes. Treatment of cells with drugs that inhibit ATM and CHEK2 activity showed that tumor cells are more sensitive to the inhibition of these proteins than PHK. Besides, we show that PHK expressing HPV16 E6 alone or along with HPV16 E7 were more sensitive to these inhibitors than control PHK or PHK expressing only E7. Moreover, PHK expressing E6 mutants defective for p53 degradation were less sensitive than PHK expressing E6wt. Moreover, to potentiate the effect observed by the ATM and CHEK2 inhibition, we treated cells lines with Doxorubicin and Cisplantin. We observed that tumor cells lines and PHK expressing HPV16 E6 or HPV16 E6/E7 were more sensitive to DNA damage induction. Altogether, these results indicated that these genes are required for HPV-transformed cells survival. Besides, our results suggest that this effect is related to HPV16 E6 oncoprotein expression and its capacity to degrade p53.

Cancer is the second leading cause of death in the USA, following cardiovascular disease. Treating cancer using conventional therapies is associated with low response rates and high toxicity, because these therapies usually lack specific tumor accumulation. Loading anticancer drugs into intelligently designed polymeric nanoparticles (NPs) can serve in delivering these drugs specifically to the tumor site, thus boosting their efficacy and reducing any associated off target toxicity. Targeting NPs to the tumor site can occur through either passive or active means. In passive targeting, NPs of specific size and surface characteristics can exploit the tumor’s erratic vasculature and occluded lymphatic drainage to extravasate the systemic circulation and accumulate preferentially at the tumor site. Active targeting mandates grafting the surface of NPs with a ligand that specifically interacts with a protein expressed at higher levels at the tumor site, in comparison to elsewhere in the body. In the current research, we independently investigated the utilization of passive and active targeting strategies to treat aggressive forms of cancer. Initially, passively targeted poly(lactic-co-glycolic acid) (PLGA) NPs to treat aggressive forms of endometrial cancer (EC) were investigated. A novel combination of soluble paclitaxel (PTX), a first line chemotherapy for EC, and soluble BIBF1120 (BIBF, nintedanib), an antiangiogenic molecular inhibitor, was first tested against three EC cell lines bearing different p53 mutations. The results showed that only EC cells with loss of function (LOF) p53 were sensitive to the combination therapy, indicating the potential of this combination to engender synthetic lethality to PTX. Next, NPs loaded with PTX were investigated with respect to the impact of varying the polymer lactic acid to glycolic acid ratio and the surfactant type on the major physicochemical properties of the prepared nanoparticles, drug loading, cellular uptake, cytotoxicity, and drug release. The optimum formulation was then loaded with BIBF and the combination of independently loaded passively targeted NPs were further evaluated for in vivo activity against a xenograft model of LOF p53 EC. The combination of independently loaded NPs exhibited the highest reduction in tumor volume and prolonged survival when compared to soluble PTX, PTX NPs or untreated control. These data highlight this specific combination of NPs as a novel promising therapy for LOF p53 EC. In a second study, the use of actively targeted NPs to treat liver cancer was explored. In this study, a combination of small interfering RNA (siRNA) against astrocyte elevated gene-1 (AEG-1), and all-trans retinoic acid (ATRA) was investigated as a new therapy for hepatocellular carcinoma (HCC). AEG-1 is a highly expressed oncogene that is directly involved in HCC progression and aggressiveness, in addition to reducing the ability of retinoic acid to induce apoptosis in HCC cells. First, a new conjugate was synthesized that was capable of delivering siRNA selectively to HCC cells, using galactose as a targeting moiety. The conjugate was prepared by linking poly(amidoamine) (PAMAM) dendrimers, polyethylene glycol (PEG) and lactobionic acid (Gal, disaccharide containing galactose) (PAMAM-PEG-Gal). We confirmed the synthesis of the conjugate using 1H-NMR, Mass spectrometry and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Next, nanoplexes of the synthesized conjugate, PAMAM-PEG-Gal, and AEG-1 siRNA were prepared. Nanoplexes were further characterized for their size, surface charge, morphology, and electrophoretic mobility to identify the optimum complexation ratio between PAMAM-PEG-Gal and the siRNA. Then, mice bearing orthotopic luciferase expressing HCC cells were treated with the optimum nanoplex formulation. Results showed that a combination of AEG-1 nanoplexes and ATRA results in a significant reduction in luciferase expression, reduced liver weight, lower AEG-1 mRNA levels and increased apoptosis, when compared to utilizing nanoplexes with silencing control (siCon), siCon+ATRA, or AEG-1 nanoplexes alone. The results indicate that the combination of liver-targeted AEG-1 nanoplexes and ATRA may be a potential treatment for aggressive HCC. These data place targeted NPs as a promising efficient delivery system for cancer treatment.

No description available.

Less than half of patients with epithelial ovarian cancer (EOC) survive five years following diagnosis, underscoring the imperative need for improved treatment. Many patients, including those with advanced disease, initially respond to platinum agents, which constitute the backbone of therapy. However, tumors ultimately become resistant, rendering further treatment ineffective. Additionally, the poor tolerability of these agents warrants the exploration of more targeted treatments – one such strategy is exploiting synthetic lethal genetic relationships. Recent genomic sequencing efforts have revealed that as many of half of EOCs have homologous recombination (HR) alterations. HR is a critical pathway for the repair of platinum-induced ICLs, thus compromised HR is hypothesized to explain the initial response to chemotherapy in many patients. Accordingly, women whose tumors harbor mutations in the critical HR genes, BRCA1 or BRCA2 (BRCA1/2), demonstrate improved prognosis. BRCA1/2 mutations also confer exquisite sensitivity to inhibitors of the enzyme, poly(ADP-ribose) polymerase 1 (PARPis), hence loss of BRCA1/2 and PARP1 is synthetic lethal. A number of models have been proposed to explain this synthetic lethality, yet a consensus model that accounts for the diverse cellular roles of BRCA1/2 and PARP1 has yet to be established. Delineating the precise molecular underpinnings of PARPi action in BRCA1/2-deficient cells will aid clinicians in identifying the appropriate population of women with EOC likely to benefit from PARPi treatment and provide insight into resistance mechanisms that arise in these patients. Combining this approach with retrospective analysis of PARPi clinical trials will best define the proper indication for PARPi in EOC and other human cancers.

PARP inhibitors (PARPi) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success due to the development of resistance to PARPi. BRCA-deficient cells are unable to repair DNA double strand breaks by the accurate homologous recombination repair (HR), and therefore rely on alternative DNA repair pathways for survival. We hypothesized that RAD52-mediated DNA repair mechanisms remain active and are thus protecting some PARPi-treated BRCA-deficient tumor cells from apoptosis, and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show here that RAD52 inhibitors (RAD52i) attenuated single-strand annealing (SSA) and residual HR activity in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with small molecule inhibitors or via expression of dominant-negative mutants induced an accumulation of DSBs and selective eradication of BRCA-deficient solid tumor and leukemia cells, while BRCA-proficient cells were unaffected. Parp1-/-Rad52-/- transgenic mice are healthy and indistinguishable from wild-type mice due to the presence of the BRCA-pathway, and Parp1-/-Rad52-/- mice with inducible BRCA1-deficient leukemia displayed significantly prolonged survival when compared to Parp1-/- and Rad52-/- counterparts. Finally, PARPi + RAD52i selectively targeted BRCA1-deficient solid tumors in immunodeficient mice with minimal toxicity to normal cells and tissues which are protected by the BRCA-pathway, indicating minimal side effects. In conclusion, our data indicate that combination treatment of RAD52i and PARPi will significantly improve therapeutic outcome of BRCA-deficient malignancies compared to treatment with PARPi monotherapy, while leaving healthy cells and tissues unharmed. / Biomedical Sciences

In Krebszellen induzieren Onkogene und Chemotherapie zelluläre Seneszenz. Dabei handelt es sich um einen terminalen Wachstumsarrest, bei dem durch Trimethylierung der Aminosäure Lysin an Position 9 (K9) des Histons H3 (H3K9me3) die Aktivierung S-Phase-relevanter Gene epigenetisch blockiert ist. Obwohl Therapie-induzierte Seneszenz (TIS) das Gesamtüberleben von Mäusen mit einer Lymphomerkrankung verbessert, stellt eine Elimination seneszenter Zellen aufgrund weiterhin bestehender und neu erworbener tumorigener Eigenschaften ein wichtiges therapeutisches Ziel dar. Die Arbeit zeigt anhand des transgenen Eμ-myc Mausmodells, in dem TIS in Abhängigkeit von der H3K9-Histonmethyltransferase Suv39h1 durch Chemotherapie induziert wird, dass TIS-Zellen in vitro und in vivo einer metabolischen Reprogrammierung unterliegen, die therapeutisch genutzt werden kann. TIS-kompetente Lymphome erhöhen im Gegensatz zu TIS-kompromittierten, Suv39h1- defizienten Lymphomen den Glukoseumsatz und die Produktion von ATP. Diese Umstellung des Stoffwechsels erfolgt als Antwort auf eine erhöhte Proteotoxizität, die durch Bestandteile des Seneszenz-assoziierten sekretorischen Phänotyps (SASP) hervorgerufen wird. SASP-Faktoren lösen in TIS-Zellen erhöhten Stress im endoplasmischen Retikulum (ER) aus und forcieren die Fehlfaltung von Proteinen, die nach vermehrter Ubiquitinierung durch Autophagie unter Energieverbrauch abgebaut werden. Deshalb sind stark SASP-exprimierende TIS-Lymphome im Vergleich zu genetisch durch die Inhibition des Transkriptionsfaktors NfκB veränderten und dadurch SASP-supprimierten TIS-Lymphomen empfindlich gegenüber der Inhibition des Glukosestoffwechsels oder der Blockade von Autophagie. Beides führt zur Elimination von TIS Zellen durch Caspase-12- und Caspase-3-abhängige, ER-initiierte Apoptose. Folglich erwirkt die pharmokologische Inhibition dieser veränderten Stoffwechselbedürfnisse nach TIS Induktion eine Tumorregression und ein verbessertes Gesamtüberleben in vivo. Zusammenfassend zeigen diese Ergebnisse eine katabole Stoffwechsellage seneszenter Zellen, die therapeutisch durch konzeptionell neue „synthetisch-letale“, metabolische Therapien eliminiert werden können. Damit wird erstmals in der Krebstherapie ein Tumor-selektives Seneszenzprogramm zusammen mit der Blockade von Stoffwechselwegen genutzt. / Cellular senescence is a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3) in response to oncogene activation and anticancer chemotherapy. Although therapy-induced senescence (TIS) improves long-term outcome, senescent tumor cells acquire potentially harmful characteristics. Therefore, their quantitative elimination presents a therapeutic opportunity. In this thesis the Eμ-myc transgenic mouse lymphoma model, in which TIS depends on the H3K9 histone methyltransferase Suv39h1, is used to show mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS- competent lymphomas but not TIS-incompetent Suv39h1- lymphomas displayed increased glucose turnover and higher ATP production. The thesis demonstrates that this was due to massive proteotoxic stress, which is a consequence of the enhanced production of secretory proteins - referred to as the senescence-associated secretory phenotype (SASP) - of senescent cells. Consequently, SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, for example due to the inhibition of the transcription factor NfκB, were more sensitive to blocking glycolysis or autophagy, which lead to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic liabilities upon TIS induction in vivo prompted tumor regression and improved treatment outcome further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting. Thus, this treatment approach for the first time combines the inhibition of a tumor-specific senescence program with the interference of a metabolic pathway.

G-quadruplexes are non-canonical nucleic acid secondary structures of increasing biological and medicinal interest due to their proposed physiological functions in transcription, replication, translation and telomere biology. Aberrant G4 formation and stabilisation have been linked to genome instability, cancer and other diseases. However, the specific genes and pathways involved are largely unknown, and the work within this thesis aims to investigate this. Stabilisation of G4s by small molecules can perturb G4-mediated processes and initial studies suggest that this approach has chemotherapeutic potential. I therefore also aimed to identify cell genotypes sensitive to G4-ligand treatment that may offer further therapeutic opportunities. To address these aims, I present the first unbiased genome-wide genetic screen in cells where genes were silenced via short-hairpin RNAs (shRNAs) whilst being treated with either PDS or PhenDC3, two independent G4-stabilising small molecules. I explored gene deficiencies that enhance cell death (sensitisation) or provide a growth advantage (resistance) in the presence of these G4-ligands. Additionally, I present a validation screen, comprising hits uncovered via genome-wide screening, and also the use of this in another cell line of different origin. Sensitivities were enriched in DNA replication, cell cycle, DNA damage repair, splicing and ubiquitin-mediated proteolysis proteins and pathways. Ultimately, I uncovered four synthetic lethalities BRCA1, TOP1, DDX42, GAR1, independent of cell line and ligand. These were validated with three G4-stabilising ligands (PDS, PhenDC3 and CX-5461) using an independent siRNA approach. The latter siRNA methodology was used to screen 12 PDS derivatives with improved medicinal chemistry properties and ultimately identified SA-100-128, as a lead compound. The mechanism behind synthetic lethality with G4-stabilising ligands was explored further for DDX42, which I show has in vitro affinity for both RNA- and DNA-G4s and may represent a previously unknown G4-helicase. Also within this thesis, gene deficiencies that provided a growth advantage to PDS and/or PhenDC3 as uncovered by genome-wide and focused screening were explored. These showed enrichment in transcription, chromatin and lysosome-associated genes. The resistance phenotype of three gene deficiencies, TAF1, DDX39A and ZNF217 was further supported by additional siRNA experiments. Overall, I satisfied the primary aims and established many novel synthetic lethal and resistance interactions that may represent new therapeutic possibilities. Additionally, the results expand our knowledge of G4-biology by identifying genes, functions and subcellular locations previously not known to involve or regulate G4s.

The peptidoglycan of many Gram-positive bacteria is densely functionalized with anionic glycopolymers called wall teichoic acids (WTAs). Recent studies have shown that these polymers play crucial roles in cell shape determination, regulation of cell division, and other fundamental aspects of Gram-positive bacterial physiology. Furthermore, in pathogens they are important in host infection and play key roles in antibiotic resistance. In many cases, precise mechanisms for WTA involvement in these processes have not been established. In order to better understand the roles of WTAs in the biology of the human pathogen Staphylococcus aureus, we sought to identify their interactions with other cellular pathways. By employing a transposon screen, we found that lipoteichoic acid (LTA) synthesis, D-alanylation of teichoic acids, cell wall stress sensors, CAAX-like proteases, and peptidoglycan biosynthesis were all synthetically lethal with depletion of WTAs in Staphylococcus aureus . Further investigations revealed that several genes required when WTAs were depleted were not essential when LTAs were removed. Unexpectedly, TA D-alanylation, became essential in the absence of WTAs, but not LTAs. Examination of terminal phenotypes following WTA depletion revealed that strains lacking LTA D-alanine esters died from envelope rupture during ongoing cell division whereas strains lacking LTAs were unable to form Z rings, stopped dividing, and had altered PG biosynthesis. Finally, we designed and implemented parallel, pathway-specific chemical screens to identify inhibitors that specifically kill mutants deficient in WTAs or D-alanylation of TAs. In addition to elucidating new interactions between cell envelope pathways, and establishing distinct roles LTAs and WTAs in the cell envelope of S. aureus, these experiments provide a list of potential targets and a strategy for identifying inhibitors for these targets, in compound combinations as therapeutics against antibiotic-resistant S. aureus infections.

L’identification d’interactions synthétiques létales (SL) apparait aujourd’hui comme une approche prometteuse, qui permet de cibler directement les cellules cancéreuses. Dans cette étude, nous avons utilisé la levure Saccharomyces cerevisiae en tant qu’organisme modèle simple pour cribler des mutations SL avec une drogue, l’AICAR (5-Amino-4-Imidazole CArboxamide Ribonucleoside). L’AICAR est une molécule connue pour inhiber spécifiquement la prolifération de multiples lignées cancéreuses. Ici, nous montrons que la perte d’ubiquitination de l’histone H2B ou de méthylation de l’histone H3K4 est SL avec l’AICAR. Nos résultats pointent sur l’AICAR causant une accumulation de cellules en G1 due à ses effets sur la localisation subcellulaire de la cycline Cln3, tandis que la perte d’ubiquitination d’H2B ou de méthylation de H3K4 affectent l’expression des deux autres cyclines deG1, CLN1 et CLN2. Ainsi, l’AICAR et la perte d’ubiquitination de l’histone H2B ou de méthylation del’histone H3K4 affectent les trois cyclines simultanément, conduisant à une condition connue pourêtre SL. De plus, cette interaction chemo-genetique s’est révélée être conservée chez les cellules humaines HCT116. En effet, le knock down de RNF40, ASH2L ou MLL2 conduit à une sensibilité àl’AICAR exacerbée. Or, on sait que MLL2 est muté dans de nombreux cancers, ce qui rend cette interaction SL très intéressante dans le cadre d’une approche thérapeutique. / Identifying synthetic lethal interactions has emerged as a promising new therapeutic approach that aims to directly target the cancer cells. Here, we used the yeast Saccharomyces cerevisiae as a simple eukaryotic model to screen for mutations resulting in a synthetic lethality with 5-Amino-4-ImidazoleCArboxamide Ribonucleoside (AICAR) treatment. Indeed, AICAR has been reported to specifically inhibit the proliferation of multiple cancer cell lines. Here, we found that loss of two several histone modifying enzymes, including Bre1 (histone H2B ubiquitination) and Set1 (histone H3 lysine 4methylation), greatly enhanced AICAR inhibitory effects on growth. Our results point to AICAR causing a significant accumulation of G1 cells due to its impact on Cln3 subcellular localization, whilebre1 or set1 deletion impacts on the two other G1 cyclins, by affecting CLN1 and CLN2 expression .As a consequence, AICAR and bre1/set1 deletions jointly affect all three G1 cyclins, leading to a condition that is known to result in synthetic lethality. Most importantly, these chemo-genetic synthetic interactions were conserved in human HCT116 cells. Knock-down of RNF40, ASH2L orKMT2D induced a highly significant increased sensitivity to AICAR. As KMT2D is mutated at high frequency in a variety of cancers, this synthetic lethal interaction has an interesting therapeutic potential.