Thymocyte development is a complex process that requires precise regulation of differentiation and proliferation. Basic helix-loop-helix (bHLH) transcription factors have been shown to be crucial for proper T cell development. HEB and E2A are structurally and functionally related E proteins of the bHLH family. These proteins directly regulate the expression of a number of genes essential for lymphocyte development in a lineage- and stage-specific manner. Abrogation or compromise of their function results in the manifestation of B and T cell developmental defects. Genetic and biochemical studies have provided evidence of a significant degree of functional redundancy among E proteins. The existence of compensational abilities among different E proteins has hampered the investigation and elucidation of E protein function. As such, single gene knockouts demonstrate only limited defects in lymphocyte development. Double E2A-HEB knockouts that could eliminate E protein redundancy are embryonic lethal. In addition, conventional gene knockouts are not well-suited for discerning between intrinsic and extrinsic defects caused by E protein disruption. To eliminate functional compensation and to test the T cell intrinsic roles of E proteins during thymocyte development, we developed a conditional HEB-E2A double knockout. Specifically, we employed a loxP/Lck-Cre recombinase system to drive E protein deletion during early thymocyte development. Using this approach, we were able to reveal overlapping roles for HEB and E2A in thymocyte development that had been obscured in previous single gene knockout studies. We find that simultaneous deletion of HEB and E2A results in a severe block in thymocyte development at the DN to DP stage transition. This developmental block is accompanied by a dramatic decrease in total thymic cellularity, an increase in apoptosis, and a reduction of pTα expression. These developmentally arrested thymocytes exhibit increased proliferation in vivo and dramatic expansion ex vivo in response to IL-7 signaling. Our findings suggest that E2A and HEB are not only critical for the regulation of T cell differentiation but are also necessary to retain developing thymocytes in cell cycle arrest prior to pre-TCR expression. Together, these results imply that E proteins are required to coordinate thymocyte differentiation and proliferation. / Dissertation

The t(1;19) chromosomal translocation is present in 5% of acute lymphoblastic leukemia (ALL) cases and leads to expression of the oncogenic transcription factor, E2A-PBX1. Although t(1;19) is exclusively associated with pre-B ALL in clinical cases, murine models produce myeloid or T-lymphoid leukemias, which are not representative of the clinical disease. In this work, we have advanced progress towards the development an E2A-PBX1-driven experimental leukemia model. We initially determined that lineage-negative (lin-) hematopoietic progenitors expressing E2A-PBX1 expression fail to repopulate the B-lymphoid lineage when transplanted into irradiated recipient mice. Furthermore, E2A-PBX1 expressing, lin- fetal liver progenitors (FLPs) fail to differentiate into B-lymphocytes ex vivo. The majority of E2A-PBX1-expressing FLPs manifested an immature phenotype and displayed stem cell factor (SCF)-dependency and enhanced self-renewal. Additionally, these cells retained myeloid potential upon transplantation or stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF). DNA binding was required for the differentiation block, suggesting that E2A-PBX1 target genes are incompatible with B-lineage specification. E2A-PBX1 FLPs had a stem cell like gene expression profile, including up-regulation of the leukemic transcription factors, Hoxa9 and Meis1. These findings explain why E2A-PBX1-driven bone marrow transplant models fail to generate B-lymphoid disease and suggest that future efforts in developing a model of E2A-PBX1-driven pre-B ALL leukemia should focus on expressing E2A-PBX1 subsequent to B-lymphoid commitment. In an attempt to override the B-lymphoid differentiation block, we next expressed E2A-PBX1 in primary pre-B cells. E2A-PBX1 induced an apoptotic response in pre-B cells, which was consistent with previous observations. Since pre-B ALL induction requires secondary genetic events, we attempted to abrogate these E2A-PBX1-mediated effects by modulating expression of the Cdkn2a locus. Loss of Cdkn2a through deletion or Bmi1 overexpression failed to ameliorate the apoptotic response, suggesting that E2A-PBX1 mediated apoptosis occurs independently of Cdkn2a in murine pre-B cells. However, in the absence of Cdkn2a, co-expression of constitutively active MerTK or Ras attenuated the E2A-PBX1 mediated apoptosis. Cumulatively, these results support the notion that t(1;19) occurs subsequent to B-lymphoid commitment and requires multiple secondary genetic lesions. Data presented in this thesis represents crucial initiating steps towards the development of a pre-B ALL model mediated by E2A-PBX1. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-09-03 00:09:29.299

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The E2A-PBX1 fusion gene results from the t(1;19) chromosomal translocation that is found in 25% of pre-B-cell cases of acute lymphoblastic leukemia (ALL). The resulting encoded product contains the transactivation domains of E2A, a Class I basic helix-loop-helix transcription factor, and most of PBX1. PBX1 is a major cofactor for most members of the HOX family of homeodomain proteins and is necessary for regulating the essential role that HOX proteins play in development and tissue homeostasis. We have identified an interaction between the E2A-encoded portion of E2A-PBX1 and the CREB-binding domain (KIX) of the transcriptional coactivator CBP and demonstrated a requirement for this interaction in leukemia induction. Others have shown that HOX proteins and CBP also interact directly, with resulting inhibitory effects on the DNA-binding ability of HOX proteins and on the acetylation of substrate proteins by CBP. Several publications have also identified the interaction of HOX proteins with the PBX1 portion of E2A-PBX1 and the result is a potent transcriptional activator at PBX1/HOX target sequences. In an attempt to develop a molecular model for the induction of ALL by E2A-PBX1, we hypothesize that the addition of CBP interactive peptide elements encoded by E2A to PBX1 allows E2A-PBX1 to stabilize a ternary complex involving E2A-PBX1, HOX, and CBP resulting in the deregulated expression of critical PBX1 or HOX target genes. I demonstrate using in vitro protein-protein interactions that this ternary complex involving E2A-PBX1, HOXB4 (chosen as a representative member of the HOX family), and CBP does form. This direct interaction appears to reduce transcriptional activation by E2A-PBX1/HOXB4 heterodimers from PBX1/HOX enhancer elements. I also show that this suppression of transactivation appears to involve CBP antagonism of DNA binding by E2A-PBX1/HOXB4 heterodimers. My results are consistent with the idea that E2A-PBX1 contributes to ALL induction by promoting the redistribution of CBP away from DNA sites bound by E2A-PBX1/HOXB4 heterodimers and in favour of those sites bound by E2A-PBX1 homodimers. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-29 13:57:25.324

Introduction: Notre laboratoire a précédemment établi que Hoxa9 accélérait l’apparition de leucémie de type B induite par E2A-PBX1. Une analyse par qRT-PCR a montré que les niveaux d’ARN de Flt3, une cible de Hoxa9, étaient 32 fois plus élevés dans les leucémies Hoxa9/E2A-PBX1 par rapport que dans les leucémies E2A-PBX1. Il est important de noter que l’expression aberrante de Flt3 est retrouvée dans les leucémies ALL de type B et les AML. De plus, l’activation constitutive de Flt3 est associée à un faible pronostic. Nous avons posé l’hypothèse que la maintenance/ré-initiation des leucémies de type pré-B induites par E2A-Pbx1 est associée à la présence du récepteur Flt3. Méthodes et Résultats: Premièrement, nous avons analysé par FACS la présence de Flt3 et mesuré l’expression de Flt3 par qRT-PCR des cellules E2A-PBX1 leucémiques pré-B. Nous avons montré que les cellules leucémiques E2A-PBX1 expriment l’ARNm du gène Flt3. Cependant, le récepteur n’était détectable à la surface cellulaire que dans des proportions variant de 0.3 à 28%. Deuxièmement, nous avons évalué le potentiel leucémique des fractions positive et négative pour Flt3. Toutes deux ont été capables de ré-initier la leucémie environ 20 jours après transplantation. Des analyses par FACS ont montré qu’une proportion de cellules leucémiques exprimaient Flt3, incluant même celles provenant de la fraction Flt3-. Troisièmement, une stratégie de perte de fonction de Flt3 par shARN a été mise en œuvre afin d’examiner le rôle de la voie de signalisation de Flt3 dans les cellules leucémiques E2A-PBX1. Pour ce faire, des cellules primaires leucémiques ont été infectées, soit par le shARN anti-Flt3 soit shARN contrôle, et transplantées dans des souris receveuses. Les cellules leucémiques contenant le shARN ont été capables de régénérer la leucémie. Cependant, une proportion des cellules exprimaient toujours Flt3, ce qui indique que l’efficacité des shARn n’était pas suffisante. Conclusion et Perspectives: Nos shARN ne sont pas suffisamment efficaces sur les cellules leucémiques choisies. De ce fait, nous proposons d’utiliser des cellules leucémiques moins agressives tout en réalisant le même set-up expérimental. Des transplantations dans des receveurs KO Flt3-/- seraient également requises afin de réellement étudier l’impact de la voie de signalisation Flt3 dans la ré-initiation leucémique. / Introduction: Previous work in the laboratory have established that Hoxa9 accelerated the onset of E2A-PBX1 induced B cell leukaemia. qRT-PCR analysis showed that RNA levels of HOXA9 target Flt3 was 32-fold increased in Hoxa9/E2A-PBX1 compared to E2A-PBX1 leukaemia. It is important to note that aberrant expression of Flt3 is found in both B-ALL and AML. Moreover, constitutive activation of Flt3 is associated with a poor prognosis. We hypothesized that the acceleration of E2A-PBX1 B-ALL by Hoxa9 is caused through increased Flt3 signalling. Methods and Results: First, to evaluate whether Flt3 signalling is functionally relevant for E2A-PBX1 induced leukaemia, Flt3 expression was analysed by FACS and qRT-PCR. So far, we showed that E2A-PBX1 B-ALL express FLT3 but the receptor was detected on a variable proportion of the cells, ranging from 0.3-28 %. Secondly, we evaluated the leukemic potential of Flt3 positive and negative fractions. Both reinitiate leukaemia around 20 days post-transplantation. Thirdly, a shRNA mediated knockdown strategy for Flt3 has been applied to test the relevance of Flt3 signalling on E2A-PBX1 leukemic cells. To test this, primary leukaemic cells were infected, either with the shRNA anti-Flt3 or the shRNA control, and transplanted into recipient mice. Unexpectedly, no difference was observed between the two groups of mice. Conclusion and Relevance: Our shFLT3 is not efficient enough on the chosen leukemic cells. Therefore, we propose to apply the same set-up to a less aggressive leukaemia. Moreover, transplanting cells in Flt3-/- KO mice is required to really assess the impact of Flt3 signalling.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / Los hongos filamentosos de ambientes extremos son fuentes promisorias de metabolitos secundarios de carácter novedoso. En particular, el género fúngico Pseudogymnoascus, que habita distintos ambientes fríos, entre ellos la Antártida, posee un metabolismo secundario poco explorado. En estudios previos de la cepa fúngica antártica Pseudogymnoascus sp. 131209-E2A-C5II-EB se identificaron los genes codificantes para enzimas policétido sintasa GymB, GymC, GymD y Gym722, de los cuales solo los dos primeros se expresaron fuertemente en las condiciones de cultivo ensayadas. El objetivo de este trabajo fue identificar nuevos genes codificantes para enzimas policétido sintasa en la cepa Pseudogymnoascus sp 131209-E2A-C5II-EB, e identificar las condiciones de cultivo en las cuales se induce su expresión. Para encontrar estas condiciones de cultivo adecuadas se emplearon distintos medios de cultivo (aproximación OSMAC) y el remodelador de cromatina 5-azacitidina. Por último, se realizaron las primeras actividades para, mediante la técnica de ARN de interferencia, identificar el metabolito sintetizado por la ruta de biosíntesis de la que forma parte el gen GymD. En resumen, en este trabajo se identificó un nuevo gen codificante para una enzima policétido sintasa, al cual se denominó Gym36. Por otro lado, mediante la aproximación OSMAC, se identificó que el medio PDB es donde se obtienen mayores niveles de inducción de manera generalizada de los genes estudiados (a excepción de Gym722 que se mantuvo sin expresión). La adición del remodelador de cromatina 5-azacitidina no logró inducir la expresión de los genes estudiados, pero si se observó que la adición de dimetilsulfóxido logró alterar positivamente los niveles de expresión de estos. Finalmente, por electroporación de conidias germinadas, se logró obtener una cepa transformante de la cepa en estudio con el gen GymD atenuado. Como trabajo futuro, se espera que al contar con un mayor número de cepas atenuantes se pueda identificar el metabolito sintetizado por la ruta de biosíntesis de la que forma parte el gen. / Filamentous fungi from extreme environments are a promising source of novel secondary metabolites. In particular, fungal genus Pseudogymnoascus, which inhabits different cold environments, including Antarctica, has a secondary metabolism that has been poorly explored. In previous studies of the Antarctic fungal strain Pseudogymnoascus sp. 131209-E2A-C5II-EB the genes coding for polyketide synthase enzymes GymB, GymC, GymD and Gym722 were identified, of which only the first two were strongly expressed in the tested culture conditions. The objective of this work was to identify new coding genes for polyketide synthase enzymes in the strain Pseudogymnoascus sp 131209-E2A-C5II-EB, and to identify the culture conditions in which their expression is induced. To find these suitable culture conditions, different culture media (OSMAC approach) and the 5-azacitidine chromatin remodeler were used. Finally, the first activities were carried out to identify, through the RNA interference technique, the metabolite synthesized by the biosynthesis pathway to which the GymD gene belongs. In summary, in this work it was identified a new gene coding for a polyketide synthase enzyme, named Gym36. On the other hand, through the OSMAC approach, it was found that the PDB medium is where the highest levels of induction are obtained in a generalized manner of the genes studied (with the exception of Gym722 that remained silent). The addition of the chromatin remodeler 5-azacitidine failed to induce the expression of the genes studied, but it was observed that the addition of dimethylsulfoxide was able to positively alter the expression levels of these. Finally, by electroporation of germinated conidia, it was possible to obtain a transformant strain of the studied strain with the GymD gene attenuated. As future work, it is expected that having a greater number of attenuating strains, will allow the identification of the metabolite synthesized by the biosynthesis pathway of which the gene belongs.

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La leucémie lymphoïde représente 30% de tous les cancers chez l’enfant. SCL (« Stem cell leukemia ») et LMO1/2 (« LIM only protein ») sont les oncogènes les plus fréquemment activés dans les leucémies aiguës des cellules T chez l'enfant (T-ALL). L’expression ectopique de ces deux oncoprotéines dans le thymus de souris transgéniques induit un blocage de la différenciation des cellules T suivie d’une leucémie agressive qui reproduit la maladie humaine. Afin de définir les voies génétiques qui collaborent avec ces oncogènes pour induire des leucémies T-ALL, nous avons utilisé plusieurs approches. Par une approche de gène candidat, nous avons premièrement identifié le pTalpha, un gène crucial pour la différenciation des cellules T, comme cible directe des hétérodimères E2AHEB dans les thymocytes immatures. De plus, nous avons montré que pendant la différenciation normale des thymocytes, SCL inhibe la fonction E2A et HEB et qu’un dosage entre les protéines E2A, HEB et SCL détermine l’expression du pTalpha. Deuxièmement, par l’utilisation d’une approche globale et fonctionnelle, nous avons identifié de nouveaux gènes cibles des facteurs de transcription E2A et HEB et montré que SCL et LMO1 affectent la différenciation thymocytaire au stade préleucémique en inhibant globalement l’activité transcriptionnelle des protéines E par un mécanisme dépendant de la liaison à l’ADN. De plus, nous avons découvert que les oncogènes SCL et LMO1 sont soit incapables d’inhiber totalement l’activité suppresseur de tumeur des protéines E ou agissent par une voie d’induction de la leucémie différente de la perte de fonction des protéines E. Troisièmement, nous avons trouvé que Notch1, un gène retrouvé activé dans la majorité des leucémies T-ALL chez l’enfant, opère dans la même voie génétique que le pré-TCR pour collaborer avec les oncogènes SCL et LMO1 lors du processus de leucémogénèse. De plus, cette collaboration entre des facteurs de transcription oncogéniques et des voies de signalisation normales et importantes pour la détermination de la destinée cellulaire pourraient expliquer la transformation spécifique à un type cellulaire. Quatrièmement, nous avons trouvé que les oncogènes SCL et LMO1 sont des inducteurs de sénescence au stade préleucémique. De plus, la délétion du locus INK4A/ARF, un évènement retrouvé dans la majorité des leucémies pédiatriques T-ALL associées avec une activation de SCL, collabore aves les oncogènes SCL et LMO1 dans l’induction de la leucémie. Cette collaboration entre la perte de régulateurs de la sénescence suggère qu’un contournement de la réponse de sénescence pourrait être nécessaire à la transformation. Finalement, nous avons aussi montré que l’interaction directe entre les protéines SCL et LMO1 est critique pour l’induction de la leucémie. Ces études ont donc permis d’identifier des évènements collaborateurs, ainsi que des propriétés cellulaires affectées par les oncogènes associés avec la leucémie et de façon plus générale dans le développement du cancer. / Lymphoid leukemia represents 30% of all cancers in children. SCL (Stem cell leukemia) and LMO1/2 (LIM only protein) are the most frequently activated oncogenes in children T cell acute lymphoblastic leukemia (T-ALL). Ectopic expression of the SCL and LMO1 oncogenes in the thymus of transgenic mice causes T cell differentiation arrest during the preleukemic stage followed by development of aggressive leukemia that reproduce human disease. We therefore took several approaches to decipher the genetic pathway collaborating with these oncogenes in T-ALL induction. Using a candidate approach, we first identified the pTalpha, a gene crucial for T cell differentiation, as a direct target of E2A and HEB heterodimers in immature thymocytes. Moreover, we showed that during normal thymocyte differentiation, SCL inhibits E2A and HEB function and that a dosage between E2A, HEB and SCL normally determines pTalpha gene expression. Second, using both global and functional approaches, we identified novel target genes of E2A and HEB transcription factors and showed that SCL and LMO1 impairs thymocyte differentiation at the preleukemic stage by globally inhibiting E proteins transcriptional activity through a DNA binding mechanism. Moreover, we found that SCL and LMO1 oncogenes are either not totally able to inhibit E protein tumor suppressor activity or act in a different leukemic inducing pathway than E protein loss of function. Third, we found that Notch1, a gene found activated in almost all cases of pediatric T-ALL, operate in the same genetic pathway as the pre-TCR to collaborate with the SCL and LMO1 oncogenes in leukemogenesis. Moreover, this collaboration between oncogenic transcription factors and normal signalling pathways important for cell fate determination might explain cell-type specific transformation. Fourth, we found that the SCL and LMO1 oncogenes are inducers of senescence at the preleukemic stage. Moreover, deletion of INK4A/ARF, an event found in almost all cases of SCL associated pediatric T-ALL, collaborate with SCL and LMO1 oncogenes in leukemogenesis. This collaboration with loss of senescence regulators suggests that a bypass of senescence response would be necessary for transformation. Finally, we also showed that SCL and LMO1 direct interaction is critical for leukemia induction. These studies permitted the identification of collaborating events and cellular properties affected by oncogenes associated with leukemia and more generally in cancer development.