Musashi-2 (MSI2), a member of the Musashi family of RNA-binding proteins, is thought to play a critical role in the maintenance of stem cell populations and in the formation of aggressive tumours. Multiple studies indicate that MSI2 plays an important role in the maintenance of hematopoietic stem cell (HSC) populations and recent studies in humans identify MSI2 as an independent prognostic factor for overall survival in patients with Acute Myeloid Leukemia (AML). Importantly, though correlative studies implicate MSI2 as a contributor to aggressive disease in human AML, no study to date has attempted to analyze the functional role of MSI2 in primary human AML samples. Furthermore, though MSI2 is critical for the maintenance of HSCs, the mechanisms through which MSI2 functions are unknown. The work presented in this thesis elucidates the biochemical mechanisms through which MSI2 functions and examines the functional role of MSI2 in human AML. Using a lentiviral-mediated shRNA knockdown of MSI2, I demonstrate that MSI2 is critical for the maintenance of human AML. A loss of MSI2 greatly impairs the ability of AML samples to maintain disease in a xenotransplantation assay. MSI2 is an RNA binding protein that is thought to repress the translation of target mRNAs in the cytoplasm and prevent the maturation of microRNAs (miRNAs) in the nucleus. The targets of MSI2 are believed to be potent regulators of stem-ness and dysregulation of these targets could very well contribute to neoplastic transformation. Cross-linking immunoprecipitation followed by next generation sequencing (CLIP-Seq), revealed the RNA binding properties of MSI2 and the RNA targets bound by MSI2. To identify novel MSI2 protein interactors, the MSI2 locus was endogenously tagged with the promiscuous biotin ligase BirA* and subjected to BioID analysis. When compared to appropriate controls, we were able to robustly identify proteins that associate with MSI2. The analysis of one of these protein binding partners, Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) reveals a critical role in the normal function of HSCs.

The hematopoietic system is responsible for the production of billions of mature cells everyday. These mature cells are “differentiated”, meaning that they have gone through a process that has allowed them to become specialized to perform a very specific role. Throughout the process of differentiation, most functional cells lose their ability to proliferate. The continued production of these functional cells comes from a pool of rare, quiescent, hematopoietic stem cells (HSC). These cells maintain the production of mature cells throughout the lifetime of an organism. The Musashi-2 (MSI2) protein has been identified as a protein that is critical for the normal function of HSCs. By altering the levels of the MSI2, it is possible to greatly impair or enhance the activity of HSCs. Moreover, correlative studies implicate MSI2 as a contributor to aggressive Acute Myeloid Leukemia (AML), a disease that occurs when HSCs become dysregulated. Despite its important roles in normal and abnormal hematopoiesis, very little is known about how MSI2 functions and whether it actually has a functional role in AML. We set forth to identify mechanisms through which the MSI2 protein functions and to prove that MSI2 contributes to the maintenance of human AML. We reveal that the MSI2 protein plays a critical role for the maintenance of human AML and identify novel pathways through which the protein functions. Importantly, MSI2 is known to interact with mRNA in order to alter post-transcriptional gene expression. We thoroughly characterize the RNA-binding characteristics of MSI2 and identify a plethora of MSI2 RNA targets. In an unbiased manner, we also identify a list of MSI2-protein interactors. We identify one MSI2 protein-binding partner, Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) that is preferentially expressed in the most immature fraction of HSCs and is critical for the proper function of HSCs.

Doctor of Philosophy (PhD)

Thesis