Delineation of molecular events that occur in a PKCα-KR-mediated murine model of CLL.
PhD thesis, University of Glasgow.
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Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia of the Western world, and despite the recent evolution in clinical treatment of the disease, it remains incurable. Although current therapies such as allogeneic hematopoietic stem cell (HSC) transplantation have been successfully used to treat CLL, this is an option for only a minority, as most CLL patients are diagnosed over the age of sixty and cannot withstand the harsh transplantation procedures. Combination chemotherapy, such as fludarabine and cyclophosphamide, has been shown to significantly improve response rate and prolonged remission in CLL patients, however, no improvement in overall survival has been observed. Patients eventually relapse due to re-emergence of minimal residual disease (MRD). Therefore it is critical that further clinical therapies are investigated in order to eliminate MRD, and offer hope to patients that are unresponsive to current treatments.
CLL is marked by the presence of the accumulation of long-lived mature monoclonal B cells in peripheral lymphoid organs, bone marrow and peripheral blood with the specific phenotype of CD19hi, CD5+, CD23+ and IgMlo that resist apoptosis. The in vivo accumulation of leukaemic lymphocytes is highly facilitated by interactions of CLL cells with other cells present in their microenvironment, including stromal cells and soluble factors such as IL4. Studies have established a variety of mechanisms potentially responsible for disease progression in CLL, including chromosomal abnormalities and intrinsic defects in the apoptotic machinery due to higher levels of the anti-apoptotic protein Bcl-2 family member proteins Bcl-2 and Mcl-1, thus making this disease extremely heterogeneous. Although the apoptotic machinery is certainly dysregulated in CLL, it is not simply a disease of a clonal accumulation of B cells, rather, proliferation is occurring as well as apoptosis, accounting for up to 2% of the clone size per day. CLL B cell proliferation centres exist within lymph nodes (LN) and bone marrow (BM) where B cells receive signals from their B cell antigen receptor (BCR) to proliferate, generating a very aggressive form of the disease. In addition, evidence suggests that stimulation through the BCR plays a pivotal role in pathogenesis of CLL since CLL B cells have a phenotypic profile of B cells activated by antigen interaction and a genetic expression profile of antigen experienced B cells.
During the course of our studies assessing the impact of modulating protein kinase C (PKC) signaling in B cell development in vitro or in vivo, we developed a unique model system to investigate the mechanisms underlying the induction of CLL. Introduction of full length, catalytically inactive PKCα (PKCα-KR) into HSCs derived from wild type mouse fetal liver (FL), and subsequent culture of the cells either in vitro or in vivo resulted in the generation of a population of B lymphocytes that are phenotypically similar to human CLL cells (CD19hi, CD5+, CD23+, IgMlo). PKCα-KR-expressing FL cells also expressed enhanced proliferative capacity over untransduced cells and were refractory to apoptosis. These results indicate that the subversion of PKCα signaling acts as an oncogenic trigger for developing B lymphocytes. The aim of this project was to identify similarities between our murine CLL (mCLL) model and human CLL and investigate putative translational therapeutic targets. The main findings of this study implicate PKCβII as an important survival and proliferation signal within mCLL. Cyclin D1 is also upregulated within mCLL, linked to an increase in the proliferative capacity of mCLL cells, and is regulated through transcriptional repressor 4EBP1, which appears inactive in both mCLL and human CLL. In addition, PKCα-KR transduced cells harbour the potential for lineage plasticity in a microenvironment-dependent manner, whereby PKCα-KR B cells lineage switch to T cells upon Notch ligation. The reprogramming occurs via a reduction in B cell specific genes and an upregulation of T cell specific genes, implicating the deregulation of PKCα activity/expression as a potential mechanism for lineage trans-differentiation during malignancies. Importantly, in human CLL, PKCα is downregulated at the transcript and protein levels implicating it a tumour suppressor, highlighting the translational capacity of our CLL mouse model.
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