published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Previously a t(2;14)(p13;q32) translocation was characterized in four unusually aggressive cases of B cell chronic lymphocytic leukemia (B-CLL). A gene located near the 2p13 breakpoint, B cell lymphoma/leukemia 11A (BCL11A), was shown to overexpress 3 isoforms (BCL11A-XL, L and S). Bcl11a knockout mice are severely impaired in B cell development at the early (pro-B) stage. I have further characterized BCL11A, focusing on the most abundant and evolutionarily conserved isoform, BCL11A-XL (XL). I demonstrated that XL resides in the nuclear matrix, is modified by ubiquitination, and is destabilized by B cell antigen receptor ligation. I identified domains within XL required for its localization within nuclear paraspeckles and for its transcriptional repression. While BCL11A-XL represses model promoters in non-B cells, its biologically relevant targets in B lymphocytes were unknown. I have identified and confirmed a number of XL targets which are both up- and down-regulated by XL over-expression in B cell lines. A number of these genes have been implicated in B cell function, including the V(D)J recombination activating (RAG) genes. Both RAG1 and RAG2 transcripts were up-regulated by XL. XL binds to the RAG1 promoter and RAG enhancer (Erag) in vivo as well as in vitro. Unexpectedly, XL repressed RAG1 transcription in non-B cells, indicating that additional B cell-specific factors are required for activation. Overexpression of XL in a V(D)J recombination-competent pre-B cell line markedly induced RAG expression and VDJ recombination. IRF4 and IRF8, transcription factors previously shown to be required for early B cell development, were also induced by BCL11A-XL. I propose that the early B cell progenitor block in Bcl11a knockout mice is, at least in part, a direct result of BCL11A-XL regulation of V(D)J recombination. Further experiments are required to establish how other XL targets promote B cell lineage development and how malignant transformation such as in B-CLL may corrupt BCL11A function.

published_or_final_version / Pathology / Master / Master of Philosophy

T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid disorder that results from an over proliferation of immature lymphocytes in the blood and bone marrow. It has been determined that 60% of patients stricken with T-ALL aberrantly express TAL-1 and have been shown to respond poorly to chemotherapy. This research sought to determine if TAL-1 influences the expression of the Bcl-2 family members Bcl-2 (anti-apoptotic), Bad and Bax (pro-apoptotic). TAL-1 and Bcl-2 levels were elevated while Bad and Bax levels were lower in etoposide-treated Jurkat cells as compared to TRAIL-treated and dual-treated Jurkat cells in which TAL-1 and Bcl-2 levels were lower while Bad and Bax levels were elevated. These results suggest TAL-1 up-regulates Bcl-2 and suppress Bad and Bax expression in response to etoposide treatment, thus inducing an anti-apoptotic response in the cell. These results also suggest that TRAIL and the dual treatment of etoposide and TRAIL down-regulates TAL-1 and Bcl-2 expression while up-regulating Bad and Bax, thus inducing a pro-apoptotic response in the cell. / Department of Biology

Acute leukemias are the main type of malignancy affecting children. They are defined by their precursor cell lineage: myeloid lineage for acute myeloid leukemia (AML) and lymphoid lineage for acute lymphoblastic leukemia (ALL). In this thesis, we use systems biology approaches to characterize transcription factor (TF) programs that define novel AML subtypes. We combine this approach with machine learning methods to group patients sharing similar TF programs and risk-stratify them. We identify a 9-cluster solution with statistically significant survival differences ranging from 84% for the best group to 41% for the worst. Each of the clusters is composed of patients with various cytogenetic aberrations that would not necessarily have been classified together. We identify top aberrantly activated TFs and potential master regulators or drug targets in each cluster. We also propose a novel stratification for FLT3-ITD patients with no other cytogenetic abnormalities. These patients are currently all classified as high-risk; however, we find a low-risk subtype and identify a TF signature that is predictive of risk in this subtype. Finally, we develop a binary classifier that is able to stratify the patients into two risk groups. We find that the activity of a large cluster of HOXA TFs is highly correlated with poor prognosis. In the second part, we characterize some mechanisms of relapse in B-ALL at a single-cell resolution focusing again on the patterns of activation and deactivation of TF activity in the course of the disease in matched trios of samples (diagnosis, remission and relapse). After a discussion on some of the technical aspects of differentiating normal cells and leukemic cells at a single-cell RNA sequencing resolution, we perform computational pseudo-lineage reconstruction based on groups of TFs whose activities rise and fall together through pseudotime. We find that each patient has unique mechanisms at the earliest pseudotimes but they seem to converge at the later pseudotimes into signatures in which the B-cell identity (in the case of B-ALL) gradually fades away. We also identify small populations of cells isolated at diagnosis in the later pseudotimes which is consistent with the view that many of the persistent cells in ALL pre-exist the malignancy and are selected by the treatment. This novel systems biology approach for characterizing clinical outcome in patients and defining lineage reconstruction identifies biochemical mechanisms and signaling pathways that are responsible for the development and maintenance of the malignancy and identifies potential therapeutic targets. The results exposed in this thesis will lead to a better understanding of some of the inner workings of pediatric acute leukemias and may lead to the development of improved targeted therapies.