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"ROLE OF MUTATION/DELETION IN NZB miR-15a/16-1 LOCI IN MICRORNA PROCESSING AND DEVELOPMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)"

by
Siddha Kasar
Molecular Pathology and Immunology Program
M.S. 2008, University of Mumbai, India
B.S. 2006, University of Mumbai, India


Thesis Advisor: Elizabeth S. Raveche, Ph.D.
Professor
Department of Pathology and Laboratory Medicine

Monday, February 11, 2013
9:30 A.M., MSB Room B-610


Abstract

This thesis examines the role of miR-15a/16-1 in the pathogenesis of Chronic Lymphocytic Leukemia (CLL) and its implications for therapeutic intervention. At 4.2 per 100,000 people, CLL is the most common lymphoid malignancy in the Western World. Frequent deletion of the 13q14 region encoding miR-15a/16-1 (approximately 50-60% of CLL patients) and the associated anti-apoptotic phenotype underscores the need for further analysis of this locus in CLL.
In this thesis I have employed NZB, the de novo mouse model of CLL. NZB mice share multiple characteristics with CLL patients, including a mutation and deletion in the miR-15a/16-1 locus. The 3 flanking region of miR-16-1 possesses a T->A point mutation (syntenic with C->T in CLL patients) and a point deletion. Alterations in the 13q14 region in humans and 14q32 region of NZB mice are associated with a 50% reduction in the expression of mature miR-15a/16-1. However, it is currently unknown whether the mutation and deletion is causative for the reduced expression and CLL development.
miR-15a/16-1 targets critical cell cycle and apoptosis regulators such as Cyclin D1 and Bcl2 respectively. Initial in vitro proof-of-principle experiments have shown that overexpression of miR-15a/16-1 with microRNA mimics has a growth inhibitory effect. However, these studies were transient and have limited pre-clinical value. This thesis demonstrates for the first time, the use of systemic lentiviral delivery of miR-15a/16-1 for CLL therapy. In addition to direct exogenous delivery of miR-15a/16-1, I have also explored other avenues for increasing miR-15a/16-1 expression. In doing this I have also uncovered the role of B cell Specific Activator Protein (BSAP) and Histone deacetylases (HDAC) in the regulation of miR-15a/16-1 expression. The other major part of this thesis is focused on characterization of the NZB miR-15a/16-1 loci in relation to microRNA processing and CLL development.
During the course of this thesis I developed an ES cell construct for targeted knock-in of the NZB loci and two congenic strains of mice (wild type mouse with NZB loci and vice-versa). In addition to the findings presented in this thesis, the ES cell targeting construct and congenic mice have the potential to serve as important tools for future research.
In summary I have shown that the NZB loci lead to impaired processing of miR-15a/16-1 and B-1 expansion. However, it is possible to over-ride this block using different strategies described in this thesis.


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