Acute myeloid leukemia (AML) is a clonal malignant disease characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature blast cells. With the success of All Trans Retinoic acid (ATRA) in acute promyelocytic leukemia (AML3), differentiation therapy has become a very attractive treatment option. Ligation of CD44 (a cell surface antigen) with anti-CD44 monoclonal antibodies (mAbs) is reported to reverse the blockage of differentiation and suppress the proliferation of blasts derived from most AML subtypes. However, the molecular mechanisms underlying this apparent ‘normalization’ (reversal) of AML cells induced by CD44 have not been fully elucidated. To expand our understanding of the cellular regulation and circuitry involved, we aimed to apply a quantitative phosphoproteomic approach using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to monitor dynamic changes of phosphorylation states in HL60 cells following treatment with CD44-mAbs. Phosphoproteomic analysis identified differentially phosphorylated proteins among CD44-mAb treated and control HL60 cells that are involved in a number of major signaling pathways as determined by the Ingenuity Pathway analysis (IPA®) platform. Among others, Rho signaling emerged as a major pathway significantly changed by CD44-mAb treatment. Rho GTPases are well-recognized regulators of the actin cytoskeleton but have also been implicated in diverse cellular events such as cell polarity, microtubule dynamics, membrane trafficking, transcriptional regulation, cell growth control and development. An interesting Rho family member, PAK2 was identified in our search. PAK2 is a ubiquitously expressed serine/threonine protein kinase, which is a direct target for small GTPases and has been identified as a switch between cell survival and cell death signaling depending on its mode of activation. Western-blot analyses of cell lysates of CD44-mAb treated and control HL60 cells confirmed that the phosphorylation of PAK2 ,as well as protein level,were altered as early as 5 minutes following treatment. PAK2 knockdown decreased the effect of CD44-mAb induction of proliferation and inhibition of proliferation proving its importance for mediating it’s signaling transduction. PAK1, a structural homologue of PAK2 had the opposite effect of augmenting CD44-mAb effects suggesting a different mechanism involved. This specificity is attributed to the specific mode of activation that PAK2 exhibits which is not shared with the rest of PAK group I members. Caspase-mediated cleavage of PAK2 producing pro-apoptotic fragments is hypothesized to be the signaling transduction mediated by CD44-mAb. In-Vivo experiments show that PAK2 is essential for leukemic cell migration to the spleen. Additionally, it proved essential for CD44-mAb inhibition of leukemic cells migration to the spleen. Further validation and characterization of PAK2’s activation mode, phosphorylation dynamics, subcellular localization as well as its role in invivo migration are essential in understanding its role in AML.

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Literature on psychological aspects of childhood cancer has treated adolescents as a homogeneous group, while revealing little about their individuality. This study's purpose was to systematically explore similarities and differences in adolescent boys with leukemia and to explore nurses' perceptions of the boys. Participants were recruited from a pediatric oncology clinic; the boys were 13, 14, and 18 years old and were selected based on age, active treatment for leukemia, and rapport with the investigator. To elicit constructs used by each boy to interpret feelings, the study employed a variant of psychologist George Kelly's technique for eliciting unique organizing principles (personal constructs) by which Kelly theorized people interpret experience (1955). The boys rated themselves on their personal constructs; their nurses also rated them on the constructs. Results reveal distinctive differences and certain similarities in the boys' personal constructs. Nurses' ratings of each patient differ in varying degrees from his own.

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