In Vivo Functional Dissection of the Chromosome 13q14 Tumor Suppressor Locus Reveals That Extent of Deletions Impacts Disease Course and Phenotype

Abstract 468

Deletion of chromosomal region 13q14 represents the most common genetic aberration in chronic lymphocytic leukemia (CLL) and occurs with less frequency in other lymphoid malignancies. 13q14 deletions are commonly large and heterogeneous in size, affecting multiple genes. We recently demonstrated that the targeted deletion in mice of miR-15a/16-1 alone causes lymphoproliferations and recapitulates the spectrum of CLL-associated phenotypes in humans. However, both the penetrance and severity of the disease increased markedly upon deletion of the 110 kilobase (kb)-long minimal deleted region (MDR) that encompasses the DLEU2/miR-15a/16-1 cluster (Klein et al., Cancer Cell, 2010). Thus, we investigate here the extent to which the additional deletion of genetic material encoded in the 690 kb large genomic region telomeric of the MDR, including the DLEU7 and RNASEH2B genes, affects the phenotype and spectrum of lymphoproliferations and disease course. To this end, we generated a conditional allele that allows in vivo deletion of this 800 kb large region termed common deleted region (CDR) specifically in B cells. B-cell conditional deletion was necessary since homozygous CDR germ-line deletion led to embryonic lethality.

We demonstrate the technical feasibility of mimicking a large chromosomal deletion (800 kb) occurring in human cancer in vivo and in a cell type-specific fashion, thus establishing an animal model for this genomic aberration. We found that despite a similar time-point of disease onset (∼12 months) and disease penetrance (∼40%), the spectrum of lymphoproliferations developing in CDR-deleted mice differed from those found in MDR-deleted mice in that the fraction of CLL among the various types of B-lymphoproliferations (including CLL, CD5⁺ monoclonal B-cell lymphocytosis (MBL) and CD5^– non-Hodgkin lymphomas (NHL)) was significantly elevated. Specifically, 19/20 mice with homozygous deletion of the CDR developed lymphoproliferations that represented CLL (95% CLL; 5% MBL) vs. 12/22 of the corresponding MDR-deleted mice (55% CLL; 27% MBL; 18% NHL). Since we previously observed that MDR^+/– mice compared to wild-type mice showed a trend towards developing clonal lymphoproliferations, we established and monitored cohorts of CDR^+/– mice and wild-type littermates. 25% of CDR^+/– mice developed CD5⁺ lymphoproliferations vs. 8% of control mice. Determination of event-free survival demonstrated that CDR^+/– mice succumb to their disease earlier than wild-type littermates (P=0.0002). Compared to MDR^+/– mice, the CDR^+/– mice showed a trend towards a more aggressive disease course (70% of CDR^+/– vs. 45% of MDR^+/– mice have died at 20 months). In summary, these results suggest that the additional loss of genetic elements located telomeric of the MDR impacts the phenotype of the lymphoproliferations and potentially the severity of disease.

Notably, lymphoproliferations derived from 4 independent CLL mouse models, i.e. TCL1-tg (Yan et al., PNAS, 2006) as well as miR-15a/16-1, MDR (Klein et al., Cancer Cell, 2010) and CDR-deleted mice, expressed antibody genes with nearly identical, stereotypic antigen binding regions. Specifically, comparing all HCDR3 regions derived from MBL or CLL cases of our own (n=46) and the TCL1-tg (n=20) studies, we observed that 44% (29/66) of the junctions can be assigned to 8 sets of highly similar HCDR3 regions among the sequence collection, 5 of which have previously been defined by Yan et al. These findings provide a rationale for defining clusters of stereotypic antigen receptors in CLL-prone mice. They also suggest that genetic aberrations disrupting the control of cell growth and survival and chronic antigen-stimulation co-operate in the clonal expansion of CLL tumor cells.

In summary, published and the present results suggest that whereas deletion of the miR-15a/16-1 cluster is the critical mechanism in the pathogenesis of CLL with 13q14 aberrations, the additional loss of genetic elements encoded in the 13q14 tumor suppressor locus can significantly influence the penetrance of the phenotype, the spectrum of the lymphoproliferations, and the severity of the disease course. Since 13q14 deletions in humans are usually heterogeneous, these findings provide a clear rationale for determining the pathogenic role of the various genetic elements affected by deletions of the 13q14 locus.