<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.

Human leukocyte antigen (HLA)-mismatched haematopoietic stem cell transplantation (HSCT) represents an important therapeutic option for patients lacking suitable donors. Delayed posttransplant immune recovery constitutes one of its major complications and is most pronounced in the T cellular compartment. A novel strategy to promote de novo thymopoiesis from donor derived HSCs and to accelerate T cellular reconstitution in patients after HSCT consists in the adoptive transfer of in vitro generated T cell progenitor cells. Identification of Notch1 as the key regulator of early T-lineage development has allowed the generation of Notch ligand-based culture systems, which provide a powerful tool to generate T-lymphoid progenitors in vitro. The efficacy of murine T-lymphoid progenitors to promote T cell reconstitution has been well demonstrated in conventional mouse models. In consistency, in vitro-generated human T cell progenitors were demonstrated to promote thymic recovery in humanized mice. Yet, positive effects of in vitro generated human T cell precursors on peripheral T cell reconstitution have not been demonstrated. Moreover currently used Notch-based co-culture systems consist of genetically modified murine cell lines. With view to establishing a clinically applicable system, feeder-cell-free Notch-ligand culture systems for the generation of T-lymphopoietic progenitors are warranted. During my PhD project I developed a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Exposure of human CD34+ cord blood cells to immobilized DL-4 enabled the in vitro generation of high number of T cell progenitors, which harboured the phenotype of immature early thymic progenitor cells (ETP) and prothymocytes (proT). ETP and proT cell generated during DL-4 culture upregulated essential genes involved in early T-lymphoid development (i.e. IL7Rα, PTα, RAG1 and BCL11b) and had undergone stage-specific recombination of the T cell receptor (TCR) locus in a similar way as in native human thymopoiesis. In limiting dilution analysis after secondary OP9/DL-1 co-culture, DL-4 progenitors displayed a highly increased T-lymphoid potential, which could be entirely attributed to the ETP and proT subset. When transferred into NOD/SCID/γc-/- mice, DL-4 primed T cell progenitors migrated to the thymus and accelerated intrathymic T cell differentiation and emergence of functional, mature and polyclonal αβ T cells in the periphery. In a co-transplantation approach, which more closely mimics a clinical setting, DL-4 progenitors and untreated CD34+ cells from HLA-disparate donors were simultaneously injected in the same recipient. This procedure allowed even more rapid and more robust T cell reconstitution. HLA-tracking of the distinct graft sources further showed, that DL-4 progenitors specifically reconstituted the T-lymphoid compartments. This work provides further evidence for the ability of in vitro-generated human T cell progenitors to promote de novo thymopoiesis and shows for the first time, that these cells accelerate peripheral T cell reconstitution in humanized mice. The availability of the efficient feeder-cell-free DL-4 culture technique represents an important step towards the future clinical exploitation translation of in vitro generated T-lymphoid progenitor cells to improve posttransplant immune reconstitution

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

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La leucémie lymphoblastique aiguë à cellules T (LLA-T) figure parmi les cancers infantiles les plus fréquents, représentant environ 15 % des cas de leucémie lymphoblastique aiguë pédiatrique et 25 % des cas chez les adultes. Cette maladie se caractérise par un blocage de la différenciation dans la lignée T et une accumulation de lymphoblastes non fonctionnels. Au cours des dernières décennies, les taux de survie se sont améliorés grâce à des protocoles de chimiothérapie agressifs. Cependant, ce traitement est associé à des effets secondaires graves qui affectent la qualité de vie des patients et imposent un fardeau important à leurs familles. De plus, les rechutes présentent un mauvais pronostic. Des travaux antécédents au laboratoire ont montré comment les oncogènes SCL et LMO1 reprogramment les thymocytes immatures au stade DN3 en cellules souches pré-leucémiques qui s’auto-renouvelent (pré-LSC). Les pré-LSCs sont à l'origine de la leucémie, elles sont résistantes à la chimiothérapie et causent des rechutes. Les pré-LSCs dépendent autant des oncogènes initiateurs que des signaux provenant de l'environnement thymique qui activent la voie NOTCH1/MYC. Finalement, un crible de petites molécules visant à inhiber les pré-LSCs dans leur niche a permis d’identifier le 2-MeOE2 comme un composé qui tue les pré-LSCs et diminue les niveaux de la protéine MYC. Dans cette thèse, nous avons cherché à comprendre les mécanismes de régulation de MYC dans les cellules pré-LSC, en utilisant le 2-MeOE2 comme outil pour des approches complémentaires pharmacologiques, génétiques et biochimiques. Ces stratégies nous ont amené à définir l'importance fonctionnelle d'affiner la régulation de MYC dans les cellules pré-LSC. Ainsi, la déstabilisation de MYC via une dégradation protéasomale affecte la viabilité des cellules pré-LSC, tandis qu'un MYC résistant au protéasome induit une signature génétique apoptotique et de sénescence. Ainsi, nos données expliquent l’absence de mutations MYC dans les leucémies LLA-T. Exploitant la capacité de 2-MeOE2 à réduire les niveaux de MYC, nous avons combiné ce médicament avec les principaux agents de chimiothérapie dans la LLA-T. Nos résultats démontrent que, tant dans un modèle murin de leucémie que dans des essais précliniques utilisant des échantillons primaires de leucémies humaines, 2-MeOE2 sensibilise les cellules pré-LSC et les cellules leucémiques à la chimiothérapie. Finalement, le crible de viabilité des pré-LSCs nous a permis aussi d’Identifier des inhibiteurs de la co-chaperone de type HSP90, Cdc37, qui réduisent les niveaux de DL1, le ligand de NOTCH1, exprimés par le stroma, et représentent ainsi de nouveaux régulateurs cellulaires non autonome de l'activité d'auto-renouvellement des cellules pré-LSCs. En effet, plusieurs inhibiteurs agissant sur CDC37/HSP90 in vitro et in vivo reproduisent le phénotype induit par la déplétion de Cdc37, y compris l'inhibition de l'activité des cellules pré-LSC. En conclusion, cette thèse met en lumière le potentiel de la dégradation de MYC en tant qu'approche prometteuse pour améliorer le traitement de la LLA-T en combinaison avec une chimiothérapie standard. De plus, une nouvelle stratégie ciblant le stroma thymique avec des inhibiteurs de Cdc37/Hsp90 a émergé, offrant une nouvelle approche pour inhiber les cellules pré-LSC. Ces découvertes font progresser notre compréhension de la LLA-T et ouvrent des perspectives pour des traitements plus efficaces et moins onéreux, dans le but ultime d'améliorer la qualité de vie des patients et les résultats du Traitement. / T acute lymphoblastic leukemia (T-ALL) is a common type of childhood cancer, representing approximately 15% of paediatric and 25% of adult Acute Lymphoblastic Leukemia cases. This disease is characterized by differentiation arrest in the T-lineage and excessive accumulation of non-functional lymphoblasts. During the last decades, survival rates have improved due to the intensification of chemotherapy regimens. However, we reached the limits of intensification, due to severe secondary effects that reduce the quality of life of patients and impose a burden on their families. Moreover, disease relapse is of poor outcomes. Previous work in the laboratory showed that the SCL and LMO1 oncogenes reprogram immature thymocytes at the DN3 stage into self-renewing pre-leukemic stem cells (pre-LSCs). Mechanistically, we showed that pre-LSCs depend on both the initiating oncogenes and the signals that these cells receive from the thymic microenvironment, triggering the NOTCH1/MYC pathway. Pre-LSCs give rise to leukemia, are chemoresistant and cause relapse. Previously, in a niche-based pre-LSCs inhibition screen we identified 2-MeOE2 as a compound that kills pre-LSCs and downregulated MYC. In this thesis, we aim to understand the mechanisms of regulation of MYC in pre-LSCs, using 2-MeOE2 as a tool compound for complementary pharmacological, genetic and biochemical approaches. These strategies led us to define the functional importance of fine-tuning MYC regulation in pre-LSCs. Indeed, destabilizing MYC via proteasomal degradation is lethal for pre-LSCs whereas a proteasome resistant MYC induces a senescent and apoptotic gene signature. Our results are consistent with the lack of MYC mutations in T-ALL, despite MYC essentiality. Then, we leveraged the capacity of 2-MeOE2 to decrease MYC levels and combined this drug with the main chemotherapeutic agents in T-ALL. We show that 2-MeOE2 sensitizes pre-LSCs to chemotherapy in our genetic model of leukemia and in pre-clinical models using primary human T-ALL samples xenografted in immune-deficient mice. Additionally, the niche-based viability screen identified a cluster of compounds that target the HSP90 co-chaperone CDC37, causing decreased expression of the NOTCH1 ligand, DL1 in the stroma, and therefore, decreased pre-LSC viability. Indeed, several inhibitors acting on CDC37/HSP90 both in vitro and in vivo reproduce the phenotype induced by depletion of Cdc37, including the inhibition of pre-LSC activity. Therefore, our work identified noncell autonomous inhibitors of pre-LSC self-renewal by their capacity to regulate DL1 in stroma cells. In conclusion, this thesis shed light on the potential of MYC degradation as a promising approach to enhance T-ALL treatment when combined with standard chemotherapy. Additionally, targeting the thymic stroma with CDC37/HSP90 inhibitors emerged as novel means to inhibit pre-LSCs. Collectively, these findings advance our understanding of T-ALL and open up avenues for more effective and less burdensome treatments, ultimately aiming to improve patient quality of life and treatment outcomes.