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B.S. 2004, College of Saint Elizabeth
Thesis Advisor: Elizabeth S. Raveche, Ph.D.
Department of Pathology and Laboratory Medicine
Friday, April 30, 2010
MSB C-555, 10:00 A.M.
New Zealand Black (NZB) mice serve as a de novo model for human chronic lymphocytic leukemia (CLL), in that they spontaneously develop an age-associated clonal expansion of B-1 cells. Similar to a subset of human patients, the NZB also manifest a monoclonal B-cell lymphocytosis (MBL)-like disease of pauciclonality, preceding the progression of a dominant clone to CLL. Alterations, ranging from deletions and mutations, in the 13q14 region containing microRNAs mir-15a/16-1 are present in over 50% of CLL patients, contributing to decreased levels of mature miR-15a/16. In the present studies, the syntenic region of the DNA of NZB revealed a germline point mutation in the 3’ flanking sequence of mir-16-1, nearly identical to the one reported in human CLL. This resulted in reduced levels of mature miR-15a and miR-16 in the spleen, as well as in both normal and malignant B cells. Higher levels of primary mir-15a/16-1 were present in RNA sources with the NZB mutation including artificial expression vectors in COS cells, suggesting a role for the mutation in the improper processing of mir-15a/16-1 primary transcript into mature form. Addition of miR-15a/16 led to an accumulation of cells in G1 in both primary NZB malignant B-1 cells and the in vitro NZB-derived malignant B-1 cell line. The major mechanism of action for miR-15a/16 is its binding to the 3’UTR of target mRNA, inhibiting translation. Protein levels of the cyclin D1 oncogene, a predicted miR-15a/16 target and cell cycle regulator of G1/S transition, were reduced in the NZB cell line following miR-15a/16 addition. Using a GFP lentiviral sensor system, the observed G1 arrest was found to be mediated by the direct interaction of miR-15a/16 with the cyclin D1 3’UTR. It was hypothesized that a relationship also exists between miR-15a/16 deficiency and drug resistance since miR15a/16 also targets the anti-apoptotic family of bcl proteins. The NZB-derived malignant B-1 cell line is a robust, chemo-resistant cell line, mimicking aggressive CLL. Transfection of this NZB cell line with miR-15a/16 synergized with sub-optimal doses of anti-cancer agents to augment apoptotic-induction by: nutlin, an MDM2 antagonist; genistein, a tyrosine kinase inhibitor; and a purine analog, fludarabine. Most importantly, miR-16 did not enhance drug-induced apoptosis in the non-NZB cell line with normal levels of miR-15a/16. These data suggest that the mir-15a/16-1 cluster may play an important role in therapeutic efficacy and that NZB B-1 chemoresistance may be related to reduced miR-15a/16 expression. In a subset of human CLL and in the NZB model of CLL, there is an increase in a population of cells with stem cell-like characteristics referred to as ‘side population’ (SP) based on their ability to exclude Hoechst. Sorted SP cells from NZB also had decreased levels of miR-16, but unlike the malignant B-1 cells, also had decreased miR-181a, which is associated with stem cells. Despite the fact that NZB SP cells express stem cell markers and SP B220- cells can differentiate into B-1 cells in vitro, they did not transfer disease in vivo, suggesting that NZB SP cells are not cancer stem cells. To further analyze the role of the SP in CLL, patients were analyzed for the correlation between SP and responsiveness to chemotherapy. Patients whose initial SP levels were low and did not increase during the course of treatment had a positive response to chemotherapy. In contrast, a patient who initially exhibited a large SP that further increased during therapy was unresponsive to treatment. Based on the data obtained from NZB and human patients, the SP cells may support the growth of the malignant B-1 cells. Restoration of miR-15a/16 to NZB mice in vivo using lentiviral vectors expressing GFP and the wild-type mir-15a/16-1 sequence (TW-15a/16) did not alter SP levels, but did result in a reduction of NZB disease compared to control NZB. Upon miR-15a/16 addition, aneuploid malignant B cells in the peritoneum, spleen, and blood were arrested in G1 within 7-8 days, followed by their eventual death within 28-29 days. NZB injected with TW-15a/16 also exhibited a decrease in live GFP+ B-1 cells that integrated the mir-15a/16-1, and an increase in the percentage of apoptotic GFP+ B-1 cells relative to live GFP+ B-1 cells. Histopathology revealed a marked decrease in the number of malignant lymphocytes infiltrating the red and white splenic pulps following miR-15a/16 therapy. The results from this thesis demonstrate that reduced miR-15a/16 levels are critical in the development and maintenance of malignant B-1 cells, targeting cell cycle and apoptosis regulators. Finally, restoration of miR-15a/16 proves to be a potential therapy for CLL since the malignant B cells are the predominant cells negatively impacted by the treatment.