Acute myeloid leukemia (AML) is a genetically heterogeneous disease and represents about 20% of pediatric leukemias. Survival rates vary depending on subtypes but are particularly unfavorable for acute megakaryoblastic leukemia (AMKL), a rare subtype of AML that usually affects children under 3 years old (≤ 30% survival for certain subtypes of AMKL). In pediatrics, genetic rearrangement leading to the expression of a chimeric fusion gene are present in many cases and are considered initiator events in the development of leukemia. In AMKL cases, more than 70% of them exhibit such rearrangement. Several of these chimeric transcripts, such as NUP98::KDM5A and CBFA2T3::GLIS2, occur in a higher proportion of cases. The analysis of the transcriptome from pediatric leukemic cases allowed us to identify new chimeric fusion transcripts in pediatric leukemias. Specifically, we discovered BPTF as a new fusion partner of NUP98 in the case of acute megakaryoblastic leukemia (AMKL), and the ACIN1::NUTM1 fusion in B-cell lymphoid leukemias. These studies have refined the molecular classification of these leukemias and provided tools for diagnosis and disease monitoring. The hypothesis of my thesis is that the NUP98::KDM5A and CBFA2T3::GLIS2 fusions are oncogenic and their expression in normal human hematopoietic and progenitor cells leads to transformation into acute megakaryoblastic leukemia in immunodeficient recipient mice, allowing for the generation of renewable xenograft models. My work has contributed to the generation of AMKL models with NUP98::KDM5A (N5A) and CBFA2T3::GLIS2 (CG2) fusions. To do this, we optimized a pipeline for transducing these chimeric genes in CD34+ cells isolated from cord blood, followed by transplantation into immunodeficient mice. These xenograft models phenocopy the leukemia of patients from a morphological, immunophenotypic, and transcriptomic standpoint. These synthetic AMKL models can be serially transplanted into mice and have a high frequency of leukemic stem cells. I also contributed to the development of a unique patient-derived xenograft (PDX) model derived from primary cells of a patient with an NUP98::BPTF genotype AMKL leukemia. These synthetic and PDX models then served as substrates for my experiments and those of several members of our laboratory. My research has allowed us to identify and characterize new biomarkers specific to NUP98- rearranged and CBFA2T3::GLIS2 positive AMKL. Taking advantage of the biomass generated by these AMKL leukemia models, we conducted transcriptomic and proteomic studies of the membrane surface. These results were compared to normal cells isolated from cord blood to identify several surface proteins specific to each leukemia genotype and shed light on new potential biomarkers. Furthermore, we confirmed the sensitivity of our AMKL models to JAK-STAT pathway inhibitors and performed synergy assays between JAK-STAT and the PI3K-AKT-mTOR pathway inhibitors. These experiments demonstrated the synergistic induction of apoptosis in our models upon the combine exposure to JAK-STAT and PI3K-AKT-mTOR pathway inhibitors. These works allowed us to identify potential therapeutic vulnerabilities of AMKL. Finally, since research on AMKL is affected by the limited number of patient samples, the human models and molecular data presented in this thesis constitute an invaluable resource to accelerate translational research for these high-risk leukemias. / La leucémie myéloïde aiguë (LMA) est une maladie hétérogène sur le plan génétique et représente environ 20% des leucémies pédiatriques. Les taux de survie varient selon les sous- types mais sont particulièrement défavorables pour les leucémies aiguës mégacaryoblastiques (AMKL), un sous-type rare de LMA touchant généralement les enfants de moins de 3 ans (≤ 30% de survie pour certains sous-types d’AMKL). En pédiatrie, les réarrangements génétiques entraînant l’expression d’un gène de fusion chimérique sont présentes dans un grand nombre de cas et sont considérées comme des événements initiateurs à l’origine de la leucémie. Chez les leucémies de type AMKL, c’est plus de 70% des cas qui présentent un tel réarrangement. Quelques-uns de ces transcrits chimériques, tels que NUP98::KDM5A et CBFA2T3::GLIS2, surviennent dans une plus grande proportion des cas. Dans le cadre de mes recherches, l’analyse du transcriptome de leucémies pédiatriques nous ont permis de mettre en évidence de nouveaux transcrits chimériques. Notamment, nous avons découvert BPTF comme étant un nouveau partenaire de fusion de NUP98 dans le cas d’une AMKL, ainsi que la fusion ACIN1::NUTM1 chez des leucémies lymphoïdes à cellules B. Ces travaux ont permis de raffiner la classification moléculaire de ces leucémies et propose de nouvelles approches pour le diagnostic et le suivi de la maladie. L’hypothèse de ma thèse est que les fusions NUP98::KDM5A et CBFA2T3::GLIS2 sont oncogéniques et leur expression chez des cellules souches hématopoïétiques et progénitrices humaines normales entraîne une transformation en leucémie aiguë mégacaryoblastique dans les souris receveuses immunodéficientes, permettant de générer des modèles de xénogreffe. Mes travaux ont contribué à la génération de modèles d’AMKL arborant les fusions NUP98::KDM5A ainsi que CBFA2T3::GLIS2. Pour ce faire, nous avons optimisé un processus de transduction de ces gènes chimériques chez des cellules CD34+ isolées de sang de cordon, suivi de transplantation chez la souris immunodéficiente. Ces modèles de xénogreffe récapitulent la leucémie des patients aux points de vue morphologique, immunophenotypique et transcriptomique. Ces modèles synthétiques d’AMKL peuvent être transplantés de manière sériée en souris et présentent une fréquence élevée de cellules souches leucémiques. De plus, nous avons aussi développé un modèle pdx unique (patient derived xenograft) dérivé des cellules primaires d’un patient atteint d’une leucémie AMKL présentant la fusions NUP98::BPTF. Ces modèles synthétiques et pdx ont ensuite servi de substrats à mes expériences ainsi que celles de plusieurs membres du laboratoire. Mes recherches ont permis d’identifier et de caractériser de nouveaux biomarqueurs spécifiques aux AMKL présentant un transcrit de NUP98 réarrangé et CBFA2T3::GLIS2. Tirant avantage de la biomasse générée par ces modèles de leucémie AMKL, nous avons fait des études transcriptomiques et protéomiques de la surface membranaire de nos modèles. Ces résultats furent comparés aux cellules normales isolées de sang de cordon afin d’identifier plusieurs protéines de surface spécifiques aux leucémies initiées par NUP98 réarrangé et CBFA2T3::GLIS2 afin de mettre en lumière de nouveaux biomarqueurs potentiels. De plus, nous avons aussi confirmé la sensibilité de nos modèles AMKL aux inhibiteurs de la voie JAK-STAT ainsi que démontré l’induction synergique de l’apoptose de nos modèles en présence des inhbitieurs combinés des voies JAK-STAT et PI3K-AKT-mTOR. Finalement, puisque la recherche sur les AMKL est ralentie par la quantité limitante d’échantillons de patient, les modèles humains et les données moléculaires présentés dans cette thèse constituent une ressource inestimable afin d’accélérer la recherche translationnelle pour ces leucémies à haut risque.

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The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

The aim of the work described in this thesis is to investigate the nature and mechanisms of action of repellent cues for growing axons. In particular I try to resolve the controversy in the literature regarding the need for protein synthesis in the growth cone in response to external guidance cues. My results resolve the conflicting data in the literature on Semaphorin-3A signalling, where differing labs had shown that inhibiting protein synthesis either blocks or has no effect upon repulsion. They demonstrate the presence of at least two independent pathways, protein synthesis-dependent mTOR activation and -independent GSK3? activation. The higher sensitivity of the synthesis-dependent pathway, and its redundancy at higher concentrations where synthesis-independent mechanisms can evoke a full collapse response alone, resolve the apparent conflict. My experiments also demonstrated that Nogo-?20, a domain of Nogo-A, requires local protein synthesis to cause collapse. Unlike Semaphorin-3A, the dependence of collapse upon protein synthesis is concentration-independent and does not involve guanylyl cyclase, but it does share a dependence upon mTOR activity and the synthesis of RhoA, sufficient to cause collapse downstream of Semaphorin-3A. The other axon-repelling domain of Nogo-A, Nogo-66, is partially dependent upon the proteasome instead. It does not share a common pathway with Nogo-?20, except that both are RhoA-dependent. I further attempted to identify the nature of a repulsive activity found in grey matter, ruling out a previously suggested candidate identity. Finally, I examined the phenomenon of nitric oxide-induced growth cone collapse. My experiments revealed that S-nitrosylated glutathione causes growth cone collapse through the activity of protein disulphide isomerase. This mechanism shows only a partial dependence upon soluble guanylyl cyclase, but I argue that it has total dependence upon an S-nitrosylated donor. Coupled with its apparent relation to S-palmitoylation, the reciprocal of S-nitrosylation, I propose that nitric oxide causes collapse by crossing the cell membrane to inhibit S-palmitoylation-determined localisation of proteins. These results reveal some of the many pathways involved in growth cone collapse, whose further characterisation may provide new targets for the treatment of injuries of the central nervous system.

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis. Dysregulation of mTOR signaling occurs in many human cancers and its inhibition causes arrest at the G1 cell cycle stage. However, mTOR’s impact on mitosis (M-phase) is less clear. Here, suppressing mTOR activity impacted the G2-M transition and reduced levels of M-phase kinase, Aurora A. mTOR inhibitors did not affect Aurora A mRNA levels. However, translational reporter constructs showed that mRNA containing a short, simple 5’-untranslated region (UTR), rather than a complex structure, is more responsive to mTOR inhibition. mTOR inhibitors decreased Aurora A protein amount whereas blocking proteasomal degradation rescues this phenomenon, revealing that mTOR affects Aurora A protein stability. Inhibition of protein phosphatase, PP2A, a known mTOR substrate and Aurora A partner, restored mTOR-mediated Aurora A abundance. Finally, a non-phosphorylatable Aurora A mutant was more sensitive to destruction in the presence of mTOR inhibitor. These data strongly support the notion that mTOR controls Aurora A destruction by inactivating PP2A and elevating the phosphorylation level of Ser51 in the “activation-box” of Aurora A, which dictates its sensitivity to proteasomal degradation. In summary, this study is the first to demonstrate that mTOR signaling regulates Aurora-A protein expression and stability and provides a better understanding of how mTOR regulates mitotic kinase expression and coordinates cell cycle progression. The involvement of mTOR signaling in the regulation of cell migration by its upstream activator, Rheb, was also examined. Knockdown of Rheb was found to promote F-actin reorganization and was associated with Rac1 activation and increased migration of glioma cells. Suppression of Rheb promoted platelet-derived growth factor receptor (PDGFR) expression. Pharmacological inhibition of PDGFR blocked these events. Therefore, Rheb appears to suppress tumor cell migration by inhibiting expression of growth factor receptors that in turn drive Rac1-mediate actin polymerization.