Alterations in the mir-15a/16-1 loci impairs its processing and augments B-1 expansion in de novo mouse model of chronic lymphocytic leukemia (CLL)

New Zealand Black (NZB) mice, a de novo model of CLL, share multiple characteristics with CLL patients, including decreased expression of miR-15a/16-1. We previously discovered a point mutation and deletion in the 3' flanking region of mir-16-1 of NZB and a similar mutation has been found in a small number of CLL patients. However, it was unknown whether the mutation is the cause for the reduced miR-15a/16-1 expression and CLL development. Using PCR and in vitro microRNA processing assays, we found that the NZB sequence alterations in the mir-15a/16-1 loci result in deficient processing of the precursor forms of miR-15a/16-1, in particular, we observe impaired conversion of pri-miR-15a/16-1 to premiR-15a/16-1. The in vitro data was further supported by derivation of congenic strains with replaced mir-15a/16-1 loci at one or both alleles: NZB congenic mice (N^miR+/-) and DBA congenic mice (D^miR-/-). The level of miR-15a/16-1 reflected the configuration of the mir-15a/16- 1 loci with DBA congenic mice (D^miR-/-) showing reduced miR-15a levels compared to homozygous wild-type allele, while the NZB congenic mice (N^miR+/-) showed an increase in miR-15a levels relative to homozygous mutant allele. Similar to Monoclonal B-cell Lymphocytosis (MBL), the precursor stage of the human disease, an overall expansion of the B-1 population was observed in DBA congenic mice (D^miR-/-) relative to wild-type (D^miR+/+). These studies support our hypothesis that the mutations in the mir-15a/16-1 loci are responsible for decreased expression of this regulatory microRNA leading to B-1 expansion and CLL development.