Acute Promyelocytic Leukemia is a subset of Acute Myeloid Leukemias, and is commonly associated with the presence of chromosomal translocations leading to the expression of the PML-RARA fusion protein. Less frequent cases of APL have been identified that express rare variant RARA fusion proteins, such as NPM-RARA. The presence of these fusions results in deregulated RARA signaling and response to the RARA ligand, ATRA. However, studies have indicated that loss of retinoid signaling alone is not sufficient to result in the leukemia. The goals of this thesis were to determine genes and pathways deregulated in variant APL that can serve as cooperating events in APL, through candidate pathway analysis and high-throughput gene expression profiling. Using a cell line model expressing variant fusion proteins associated with APL, we identified the deregulation of the NF-kappaB signaling pathway in APL, and describe the functional analysis of this pathway in our in vitro model. We next assessed whether promotion of survival signals could serve as a contributing factor in the accumulation of leukemic blasts in APL patient bone marrow. Our results indicated that PML-RARA and NPM-RARA expressing cells showed increased survival in the presence of TNFalpha, compared to wild type control cells. These data suggested a greater ability on the part of NPM-RARA cells to survive in the presence of TNFalpha. We also report for the first time the gene expression profiles of, and transcriptional effects of ATRA on, cells expressing the variant fusion proteins. Finally, we determined that the partner protein Nucleophosmin (NPM) has increased cytoplasmic localization in cells expressing the APL fusion proteins, and interacts within complexes comprising of RARA and RXRA. We further determined that the fusion can interfere with NPM function and cellular localization. Taken together, these studies provide evidence for the involvement of secondary hits in APL biology.

Acute Promyelocytic Leukemia is a subset of Acute Myeloid Leukemias, and is commonly associated with the presence of chromosomal translocations leading to the expression of the PML-RARA fusion protein. Less frequent cases of APL have been identified that express rare variant RARA fusion proteins, such as NPM-RARA. The presence of these fusions results in deregulated RARA signaling and response to the RARA ligand, ATRA. However, studies have indicated that loss of retinoid signaling alone is not sufficient to result in the leukemia. The goals of this thesis were to determine genes and pathways deregulated in variant APL that can serve as cooperating events in APL, through candidate pathway analysis and high-throughput gene expression profiling. Using a cell line model expressing variant fusion proteins associated with APL, we identified the deregulation of the NF-kappaB signaling pathway in APL, and describe the functional analysis of this pathway in our in vitro model. We next assessed whether promotion of survival signals could serve as a contributing factor in the accumulation of leukemic blasts in APL patient bone marrow. Our results indicated that PML-RARA and NPM-RARA expressing cells showed increased survival in the presence of TNFalpha, compared to wild type control cells. These data suggested a greater ability on the part of NPM-RARA cells to survive in the presence of TNFalpha. We also report for the first time the gene expression profiles of, and transcriptional effects of ATRA on, cells expressing the variant fusion proteins. Finally, we determined that the partner protein Nucleophosmin (NPM) has increased cytoplasmic localization in cells expressing the APL fusion proteins, and interacts within complexes comprising of RARA and RXRA. We further determined that the fusion can interfere with NPM function and cellular localization. Taken together, these studies provide evidence for the involvement of secondary hits in APL biology.

Tumour initiation and progression occur due to oncogenic mutations that also contribute to therapeutic resistance in many human tumours. Mutations activating the "PI3K/AKT" signalling pathway and inactivating the "TP53" tumour suppressor gene are common mechanisms that cancer cells require to proliferate and escape pre-programmed cell death. p53 mutant (p53mut) tumours not only fail to respond to DNA damaging therapy, but are also described to promote therapeutic resistance by dominant negative suppression of p53 dependent promoter activity. Our work identifies the crucial interaction between the PI3K/AKT pathway and p53 mutations that regulate treatment sensitivity in tumours. Using a combination of in vitro and in vivo techniques we demonstrate that AKT inhibition promotes reduced cellular levels of p53mut via a novel Nucleophosmin 1 (NPM) mediated regulation of the tumour suppressor p14ARF and promotes re-engagement of cell cycle arrest, senescence and increased sensitivity to ionising radiation in both in vivo and in vitro systems. We show that the PI3K/AKT pathway plays an important role in the regulation of p53mut and inhibitors of this pathway can re-sensitise treatment resistant tumours. This has helped us to simultaneously highlight the cohort of patients where the greatest efficacy may be achieved in clinical practise. We further show that the AKT mediated regulation of NPM that we describe in solid tumours is relevant in Acute Myeloid Leukaemia (AML) with mutated NPM, albeit showing physiologically different effects. This further highlights the necessity for rational treatment planning with the newer targeted agents that inhibit specific signalling pathways in AML patients.

La Nucléophosmine (NPM1/B23) est une petite chaperonne moléculaire impliquée dans de nombreux processus cellulaires, tels que la régulation de l’expression génique ou le contrôle du cycle cellulaire. De nombreuses études rapportent une surexpression de NPM1 dans divers types de tumeurs solides incluant les cancers de la prostate, et son rôle proH prolifératif dans des lignées cellulaires tumorales d’origines variées est bien établi. La première partie de notre travail s’est attaché à évaluer le potentiel oncogénique de NPM1 dans l’épithélium prostatique in vivo. Pour cela, nous avons généré un modèle de souris transgéniques qui surexpriment NPM1 spécifiquement dans l’épithélium de la prostate. Ces animaux présentent une hyperplasie prostatique associée à une augmentation de l’index prolifératif de l’épithélium. Nos expériences révèlent que NPM1 pourrait lever la quiescence des cellules épithéliales différenciées en dérégulant l’expression de gènes clés de la régulation du cycle cellulaire, comme la Cycline E ou p27kip1. Bien que ces souris ne développent pas de lésions néoplasiques, ces données suggèrent que NPM1 participe à la carcinogenèse prostatique en association avec d’autres lésions oncogéniques. La seconde partie du travail visait à comprendre la nature des mécanismes qui supportent la surexpression de NPM1 dans les tumeurs prostatiques. Des données récentes de la littérature indiquent un enrichissement de la protéine kinase mTOR au niveau du promoteur proximal de NPM1 dans des foies de souris. Pour déterminer s’il existe un lien fonctionnel entre mTOR et NPM1, nous avons tiré parti d’un modèle de fibroblastes embryonnaires de souris invalidés pour le suppresseur de tumeur PTEN dont l’inactivation mène à une hyperactivité de mTOR. Dans ce contexte, les taux d’ARNm et de protéines NPM1 sont augmentés par rapport aux cellules sauvages. Nos résultats montrent également que mTOR contrôle l’expression de NPM1 i) en se fixant sur son promoteur et en stimulant l’expression du gène et ii) en stabilisant l’ARNm de NPM1. Nous avons confirmé le lien entre NPM1 et mTOR in vivo grâce à notre modèle de souris invalidées pour PTEN dans l’épithélium prostatique. Enfin, nous avons montré que l’expression de NPM1 est nécessaire pour transduire les effets prolifératifs de la voie PI3K/AKT/mTOR. Ces données placent donc NPM1 comme nouvel effecteur en aval de cette voie de signalisation, faisant de cette protéine une potentielle cible thérapeutique dans les tumeurs présentant une perte de PTEN. / Nucleophosmin (NPM1/B23) is a small molecular chaperone involved in a large array of cellular processes, including the regulation of gene expression and the control of the cell cycle. Several studies have reported the overexpression of NPM1 in solid tumors from various histological origin, including prostate cancer, and its proliferative impact on several human cancer cell line is being well described. The first part of our work aimed at assessing the NPM1 oncogenic properties in the prostate gland in vivo. To do so, we generated a new transgenic mouse model that overexpresses NPM1 specifically in the prostatic epithelium. These mice harbor prostatic hyperplasia associated with an increase of the ki67 proliferative index. Our molecular investigations revealed that NPM1 could be an inhibitor of the quiescent state of epithelial cells through a dysregulation of key cell-cycle controlers such as Cyclin E or p27kip1. Although these mice do not develop neoplastic lesions, our data suggest that NPM1 overexpression accelerate prostate cancer progression when associated with other oncogenic alterations. The second part of the work aimed at understanding the mechanisms underlying NPM1 overexpression in prostate tumors. The serine/threonine Kinase mTOR was recently shown to bind to the proximal promoter of NPM1 in the mouse liver. In order to characterize a fonctionnal link between NPM1 and mTOR, we took advantage of murine embryonic fibroblast (MEF) deleted for PTEN, since these cells display a constitutive mTOR activity. In such cells, NPM1 protein and mRNA levels are increased compared to wild type MEF. We also demonstrated that mTOR controls NPM1 expression i) through its binding to NPM1 promoter, thus stimulating NPM1 gene expression and ii) by stabilizing NPM1 mRNA. We have confirmed the functional link between NPM1 and mTOR in vivo in a mouse model deleted for PTEN specifically in the prostatic epithelium. Finally, we have shown that NPM1 expression is necessary for the proliferation of PTEN knock-out MEF. These data set NPM1 as a new downstream effector of the PI3K/AKT/mTOR pathway, and suggest that it could be a new potential therapeutic target in PTEN negative human prostate cancer.

C-terminal mutations of the phosphoprotein nucleophosmin (NPM) are the most frequent genetic aberration detected in adult acute myeloid leukemia (AML). I focused on characterization of type A, B and E of AML-related C-terminal mutations. The plasmids bearing fluorescently labeled wild type or mutated NPM have been constructed to characterize mutation-induced changes in the localization of NPM. Mammalian cell lines HEK293T, HeLa and NIH 3T3 were used for production of the chimeric proteins. The intracellular localization of the mutated forms of NPM was analyzed by immunofluorescence staining and fluorescence microscopy of the living cells. The localization of the mutNPM type A and B was almost identical and predominantly cytoplasmic, while mutNPM type E was detected in nucleolus and cytoplasm simultaneously. However localization of the mutated forms was greatly influenced by the used cell line. It has been demonstrated that the exogenous NPM interacts with the endogenous NPM and that they mutually affect their intracellular localization due to heterooligomer formation. Detailed analysis of the relationship between the C-terminal mutations and the localization of the mutated NPM improves understanding of specific mutation effect on the formation and progression of AML and also specifies its prognostic...

Interferon Regulatory Factor -1 (IRF-1) is a transcription factor that acts as a tumour suppressor in cancer cells. The inactivation or deletion of IRF-1 either in one or both allele has been frequently reported in leukaemia and myelodysplasia (MDS). On the other hand nucleophosmin (NPM), a nucleo-cytoplasmic shuttling phosphoprotein is also known to be aberrant in some form of leukaemia. NPM was first proposed as a binding partner of IRF-1 in 1997 and suggested to inactivate IRF-1 by inhibiting its DNA binding ability. No further researches on the interaction between IRF-1 and NPM1 was reported prior to the start of my PhD. In the research presented here the interaction and mechanism by which IRF-1 might be inactivated by NPM was studied. Under the context of both NPM and IRF-1 being frequently associated with leukaemia and MDS, the study was done to determine the role of NPM under its naïve condition and a most frequent mutated condition (NPMc+), where the C-terminal of NPM was frequently mutated to give rise to a cytoplasmic NPM in certain leukaemia. In this current research, the direct interaction between IRF1 and NPM was further confirmed both in vitro as well as within the cells. Following this, the effect of this interaction in respect to the leukaemic condition having NPMc+ mutation was done, by comparing the end results on AML2 (leukaemic cells with intact wild type NPM) and AML3 (leukaemic cells having a single NPM allele mutated to form NPMc+) cells. In this research, overexpression of wild type NPM (NPMwt) was found to increase IRF-1 transcriptional activity. On further analysis, the DNA binding activity of IRF-1 due to the presence of NPMwt or NPMc+ was not always inhibited, instead it shows a change in binding specificity, where NPMwt bound IRF-1, lacks DNA binding ability and DNA bound IRF-1 has a reduced binding towards NPM. This is being studied further in terms of NPM overexpression and increased IRF-1 transcriptional activity, as the order of addition (order of interaction in vivo) plays a major role in activating or deactivating IRF-1. This along with the increased transcriptional activity of IRF-1 suggests a novel function of NPM, where it could act in favour of IRF- 1 activity. Additionally, the NPM induced change in IRF-1 localisation was confirmed by the cytoplasmic localised IRF-1 in NPMc+ expressing cells and nucleolar sequestration in NPMwt overexpressing cells. This gives a novel mechanism by which NPM regulates IRF-1. Further, the NPMc+ specific colocalisation of IRF-1 urges to study the other proteins that may have been re-localised in AML cells due to the NPMc+ specific interaction. A mass spectrometric analysis on the cellular distribution of total proteins were analysed between AML2 (cells with NPMwt) and AML3 (cells containing NPMc+). Specific proteins related to cancer have been identified to be differentially distributed rather than being a random translocation. With this being said, a peptide phage display technology coupled with next generation sequencing was done to identify NPMwt binding peptides that can be used in drug discovery process or as small molecule inhibitors or activators. Three different peptides were selected at the end of the study that bind very effectively to NPMwt. These peptide can either aid or restrict NPM activity and need to be validated and studied in the future.

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Many inflammatory diseases are growing increasing common in the aging society of Taiwan. Inflammation cascades can cause diseases such as rheumatoid arthritis, osteoarthritis, chronic asthma, multiple sclerosis, and so on. The clinically used anti-inflammatory drugs have many side effects and are expensive. Therefore, it is imperative that we find alternatives to these drugs. Marine natural compounds offer great hope in the development of drugs for treating inflammatory diseases. In the present study, we found that Chao-10, which is a marine-derived compound isolated from Formosan soft coral, significantly inhibited the expression of the pro-inflammatory protein, inducible nitric oxide synthase (iNOS), in the lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cell line. We suggest that Chao-10 may serve as a potential new anti-inflammatory agent. However, the mechanism by which the anti-inflammatory effects of Chao-10 are mediated is yet unclear. Therefore, we performed two-dimensional electrophoresis (2-DE) to investigate the regulatory mechanism for the anti-inflammatory effect of Chao-10. We isolated some proteins that may be involved in the anti-inflammatory mechanism of Chao-10. In addition, we used immunoprecipitation to find that nucleophosmin (NPM) could interact with nuclear factor kappa B (NF-£eB). Therefore, we hypothesize that nucleophosminmay be involved in the regulation of NF-£eB to enhance the down-regulation of iNOS proteins. In summary, the anti-inflammatory effects of Chao-10 are probably mediated through the some other signaling pathway. Importantly, Chao-10 not only offers some new biomarkers of inflammation but also provides an encouraging outlook on therapeutic approaches.

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Les thérapies ciblées constituent une révolution médicale dans le traitement du cancer. Dans ce contexte, le pseudopeptide multivalent N6L, qui cible spécifiquement les cellules tumorales et induit leur mort, apparaît comme une molécule prometteuse. Le N6L cible en effet deux nucléoprotéines surexprimées à la surface des cellules cancéreuses qui sont la nucléoline et la nucléophosmine. L'étude du mécanisme d'action anti-métastatique du N6L, dans lequel l'implication du TIMP-3 a été soulignée, a permis d'identifier une nouvelle cible : les glycosaminoglycanes sulfatés (GAG).Dans le but de développer une approche multimodale pour le diagnostic et le traitement du cancer du sein, le N6L a été greffé à la surface de nanoparticules magnétiques (NPM-N6L). La propriété de ciblage tumorale des NPM-N6L a été démontrée in vitro et in vivo. Leur cible tumorale majeure fut les GAG.Par ailleurs, l'activité anti-tumorale du N6L dans le traitement du cancer de la prostate à différents stades de la maladie a été démontrée in vitro et in vivo. La cible tumorale mise en jeu fut la nucléophosmine. En outre, ce potentiel anti-tumoral implique une diminution de l'activité du récepteur aux androgènes et probablement une voie de signalisation impliquant une interaction entre la nucléophosmine phosphorylée et le récepteur aux androgènes. / Targeted therapies constitute a revolution in the medical treatment of cancer. In this context, the multivalent pseudopeptide N6L that specifically target tumor cells and induces their death seems a promising molecule. The N6L targets two nucleoproteins that are overexpressed on the surface of cancer cells: nucleolin and nucleophosmin. The study of the N6L anti-metastatic mechanism of action in which TIMP-3 was involved, has identified a new target: sulfated glycosaminoglycans (GAG).In order to develop a multimodal approach for diagnosis and treatment of breast cancer, the N6L was grafted to the surface of magnetic nanoparticles (MNP-N6L). Tumor targeting properties of MNP-N6L was demonstrated in vitro and in vivo. Their major tumor target was GAG.Furthermore, the N6L anti-tumor activity in the treatment of prostate cancer at different stages of the disease was demonstrated in vitro and in vivo. The target involved in this activity was nucleophosmin. Furthermore, this anti-tumor potential implies a decrease in the activity of the androgen receptor and probably a signaling pathway involving an interaction between phosphorylated nucleophosmin and the androgen receptor.