Despite advances in the understanding of cancer biology, most patients with acute myeloid leukemia (AML) still die of their disease. Improving risk-stratification and identifying new targets are important steps towards personalized medicine and outcome improvement. MicroRNAs, short non-coding RNAs that hybridize to their target messenger RNAs (mRNAs) and repress the expression of the encoded proteins, are known to be involved in physiological processes like cellular differentiation, proliferation and cell survival but also play an essential role in cancer, including AML. In this thesis we demonstrated that higher expression of a single microRNA miR-181a was associated with clinical outcome in cytogenetically normal AML (CN AML) patients. In multivariable models, higher expression of miR-181a was associated with achievement of complete remission (CR), with longer disease-free (DFS) and overall survival (OS) even in consideration of other validated prognostic clinical and molecular variables. Measurement of pretreatment levels of this microRNA may improve risk-stratification for AML patients. A genome-wide gene-expression signature gave biological insights into miR-181a associated AML, and provides a basis for further functional studies. Furthermore, as higher miR-181a expression associated with improved treatment response, increasing miR-181a levels by delivering synthetic miR-181a or by agents increasing endogenous levels of this microRNA in AML blasts may represent a novel and personalized therapeutic approach in AML.

Despite advances in the understanding of cancer biology, most patients with acute myeloid leukemia (AML) still die of their disease. Improving risk-stratification and identifying new targets are important steps towards personalized medicine and outcome improvement. MicroRNAs, short non-coding RNAs that hybridize to their target messenger RNAs (mRNAs) and repress the expression of the encoded proteins, are known to be involved in physiological processes like cellular differentiation, proliferation and cell survival but also play an essential role in cancer, including AML. In this thesis we demonstrated that higher expression of a single microRNA miR-181a was associated with clinical outcome in cytogenetically normal AML (CN AML) patients. In multivariable models, higher expression of miR-181a was associated with achievement of complete remission (CR), with longer disease-free (DFS) and overall survival (OS) even in consideration of other validated prognostic clinical and molecular variables. Measurement of pretreatment levels of this microRNA may improve risk-stratification for AML patients. A genome-wide gene-expression signature gave biological insights into miR-181a associated AML, and provides a basis for further functional studies. Furthermore, as higher miR-181a expression associated with improved treatment response, increasing miR-181a levels by delivering synthetic miR-181a or by agents increasing endogenous levels of this microRNA in AML blasts may represent a novel and personalized therapeutic approach in AML.:Bibliografische Beschreibung 1 Vorbemerkung / Preliminary Remarks 2 Referat / Abstract 3 Einführung / Introduction 4 Publication 13 Zusammenfassung / Conclusion 31 Ausgewählte Publikation / Selected Publication 38 Komplette Publikationsliste / Complete List of Publications 39 Lebenslauf / Curriculum Vitae 46 Erklärung über die eigenständige Abfassung der Arbeit 50 Danksagung / Acknowledgements 51

Les cellules tueuses naturelles (NK pour Natural Killer) sont une population lymphoïde dotées d’une activité cytotoxique contre les cellules infectées ou les cellules cancéreuses. Les cellules NK ont un potentiel thérapeutique considérable en tant que thérapie cellulaire anti-tumorale, particulièrement dans le cadre des leucémies. Ces approches sont basées sur une amélioration de la production de cellules NK à partir de cellules souches hématopoïétiques (quantitative et qualitative en améliorant leur activité lytique), mais aussi sur une manipulation de la sensibilité des cellules leucémiques à la lyse par les cellules NK. L’amélioration de ces approches nécessite une compréhension plus approfondie des différents mécanismes de résistance leucémique et leur relation avec la sensibilité à la lyse. Dans ce contexte, nous avons étudié le rôle de HOXB4 dans la différenciation des cellules NK et leur fonction lytique. Nous avons montré que les cellules CD34+ différenciées en cellules NK en présence de HOXB4 ont un potentiel lytique plus important par rapport aux cellules différenciées en l’absence de HIOXB4. Cette augmentation est associée à une augmentation de la dégranulation des cellules NK en présence de cellules cibles. L’analyse transcriptionnelle globale basé sur un microréseau d'ADN montre une régulation positive de l’expression de granzyme B par HOXB4. Ces résultats démontrent que HOXB4 est un régulateur crucial dans la différenciation et la fonction des cellules NK. Ils soulignent également l’intérêt de son utilisation dans la production de cellules NK fonctionnelles dotées d’un plus grand potentiel lytique pour les stratégies d'immunothérapie anticancéreuse. Nous avons également essayé de comprendre comment l'acquisition de la résistance aux chimiothérapies par les cellules de leucémie aigüe myéloïde (LAM) influence leur reconnaissance et leur sensibilité aux cellules NK. Nous avons montré que l'acquisition de la résistance in vitro des cellules AML à la cytarabine induit une augmentation de leur susceptibilité à la cytotoxicité dépendante des cellules NK. Cette sensibilité accrue est en corrélation avec l’induction d’ULBP (UL-16 binding proteins) 1/2/3, ligands des récepteurs NKG2D, sur les cellules leucémiques résistantes. Cette induction est régulée par un mécanisme impliquant l'induction de c-Myc. Le test d’immunoprécipitation de la chromatine (ChIP) a révélé qu’ULBP1 et ULBP3 sont des cibles directes de c-Myc. L’utilisation de cellules AML primaires résistants à la chimiothérapie comme les cellules cibles, combinée à l'inhibition de c-Myc a entraîné une diminution de l'expression des ligands NKG2D et l'altération de la lyse par les cellules NK. Les propriétés d’alloréactivité des cellules NK pourraient être utilisées pour améliorer les résultats de la transplantation de cellules souches hématopoïétiques allogéniques chez les patients atteints d’AML. Cependant, la résistance croisée (chimiothérapie et NK) des blastes AML reste un problème majeur. Nous avons étudié la relation entre résistance des cellules leucémiques à la daunorubicine, la susceptibilité de ces cellules à la lyse par les cellules NK et l'expression putative des micro-RNAs. Nos résultats indiquent que l'acquisition de la résistance à la daunorubicine par les lignées de cellules parentales induit une résistance croisée à la cytotoxicité naturelle à médiation cellulaire. L'analyse des microréseaux de microRNAs a révélé que cette résistance croisée est associée à une diminution du miR-181a et une augmentation des gènes de la famille tyrosine kinase (MAP3K10 et MAP2K1) et de la famille Bcl-2 (Bcl-2 et Mcl-1). La surexpression de miR-181a dans les blastes AML entraîne l'atténuation de leur résistance à la daunorobucine et à la lyse par les cellules NK. / Natural Killer (NK) cells are a lymphoid population with potent cytotoxic activity against virus-infected or cancer cells, and which hold considerable potential for cell based therapies targeting human malignancies. Potential approaches include not only enhancing the generation of NK cells in number and improving their lytic activity, but also manipulating the susceptibility of blast cells to NK-mediated killing. Pursuing these approaches will require a more thorough understanding of the different mechanisms of resistance and their relationship with susceptibility to NK-mediated killing. In this context, we studied the role of HOXB4 in NK cells differentiation and lytic function. We showed that HOXB4 transduced MS-5 cells as compared with GFP-transduced MS-5 cells induced highly differentiated cytotoxic NK cells. This difference was associated with an increased induction of granzyme B degranulation in response to stimulation with NK cell susceptible targets. DNA microarray-based global transcriptional profiling confirmed the upregulation of granzyme B. These findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy. We attempted to elucidate how acquisition of drug resistance in AML cells influences NK cell recognition and the killing of drug-resistant blasts. We showed that the in vitro acquisition of AML cell resistance to cytarabine resulted in an increase in their susceptibility to NK-mediated cell cytotoxicity. The increased susceptibility correlates with the induction of UL-16 binding proteins (ULBP) 1/2/3 and NK group 2, member (NKG2D) ligands on target cells by a mechanism involving c-Myc induction. More importantly, chromatin immunoprecipitation assay revealed that ULBP1/3 are direct targets of c-Myc. Using drug resistant primary AML blasts as target cells, inhibition of c-Myc resulted in decreased expression of NKG2D ligands and the subsequent impairment of NK cell lysis. This study provides for the first time, the c-Myc dependent regulation of NKG2D ligands in AML. Manipulating NK-cell alloreactivity might improve outcomes after hematopoietic stem-cell transplantation; however, cross-resistance among blasts remains a drawback. We attempted to investigate the relationship between AML to daunorubicin, the susceptibility to NK cellmediated cell lysis and the putative expression of miRs. Our results indicate that the acquisition of resistance to daunorubicin by the parental cell lines resulted in the acquisition of a cross-resistance to natural cell-mediated cytotoxicity. miR microarray analysis revealed that this cross-resistance was associated with miR-181a down regulation and the subsequent regulation of the tyrosine kinase (MAP3K10 and MAP2K1) and the BCL-2 (BCL-2 andMCL-1) families. Overexpression of miR-181a in AML blasts resulted in the attenuation of their resistance to daunorobucin and to NK-cell-mediated killing.

T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR-181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV-infected individuals compared with age-and sex-matched healthy subjects. Mechanistic studies revealed that up-regulation of transcription factor ∆Np63 led to the decline of miR-181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV-infected individuals. Either reconstituting miR-181a or silencing ∆Np63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence-associated b-galactosidase (SA-β-gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV-induced T cell senescence is counterregulated by the ∆Np63-miR-181a-Sirt1 pathway. An increase of IL-2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3- effector T (Teff) cells upon manipulating the ∆Np63-miR-181a-Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR‐181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV‐infected individuals compared with age‐ and sex‐matched healthy subjects. Mechanistic studies revealed that up‐regulation of transcription factor ΔNp63 led to the decline of miR‐181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV‐infected individuals. Either reconstituting miR‐181a or silencing ΔNp63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence‐associated β‐galactosidase (SA‐β‐gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV‐induced T cell senescence is counterregulated by the ΔNp63–miR‐181a–Sirt1 pathway. An increase of IL‐2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3− effector T (Teff) cells upon manipulating the ΔNp63–miR‐181a–Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

La vaccination est largement utilisée pour la génération de lymphocytes T spécifiques contre les tumeurs. Malheureusement, cette stratégie n'est pas adaptée aux personnes âgées car leur thymus régresse avec l'âge conduisant ainsi à une baisse dans la production de cellules T et à l'accumulation de cellules immunitaires âgées ayant des défauts liés à leurs stimulations. Comme il a été démontré auparavant que L’IL-21 est capable d’induire des fonctions thymiques, nous avons émis l’hypothèse que l’injection d’IL-21 à des souris âgées stimulera la thymopoïèse. Nos résultats montrent que l’administration de l’IL-21 augmente le nombre absolu de thymocytes chez les souris âgées et augmente la migration de ces cellules vers la périphérie ou ils contribuent à la diversité du TCR. De plus les cellules T en périphérie expriment un niveau plus élevé de miR181-a, et par conséquent moins de phosphatase comme SHP2, DUSP5/6 qui inhibent le TCR. En vaccinant des souris âgées avec le peptide Trp2, les souris traitées avec l’IL-21 montrent un retard dans la croissance des cellules B16 tumorales. Cette étude montre que l’IL-21 pourrait être utilisé comme stratégie pour le rétablissement du systeme immunitaire chez les personnes âgées. / Vaccines have been largely sought for the generation of protective tumor-specific T cells. Unfortunately however, this strategy is not suitable for the elderly as their thymus regresses with age leading to a decline in T-cell production and accumulation of aged lymphocytes endowed with stimulation-related defects. Interleukin (IL)-21 has been shown to display thymostimulatory properties. Therefore, we hypothesized that IL-21 administration to ageing host may boost T-cell production and restore a competent peripheral T-cell compartment. Our study shows that IL-21 injection to aged mice lead to an increase in the thymocytes absolute number and an increase in thymocytes egress to the periphery where they enhance the T-cell receptor (TCR) diversity. Furthermore T-cell in the periphery express higher level of miR-181a and thus less TCR-inhibiting phosphatases SHP-2 and DUSP5/6 enable them to be more responsive upon stimulation. Consequently, aged rIL-21-treated mice vaccinated using a tyrosinase-related protein 2 (Trp2)-derived peptide exhibited a substantial delay in B16 tumor growth and improved survival. The results of this study highlight the immunorestorative function of rIL-21 paving its use as a strategy for the re-establishment of effective immunity in the elderly.