A Novel Mechanism for Intrachromosomal Gene Amplification in Multiple Myeloma: 1q12 Pericentromeric Heterochromatin Mediates Breakage- Fusion-Bridge Cycles of the 1q12~23 Amplicon

Abstract Gene amplification is a marked copy number (CN) increase in a restricted region of a chromosome arm, and is a mechanism for acquired drug resistance and oncogene activation. In multiple myeloma (MM), recent studies utilizing gene expression profiling, high-resolution array comparative genomic hybridization (aCGH), and global single nucleotide polymorphism arrays have all provided molecular evidence for the importance of genes in the proximal 1q. In fact, the aCGH studies have defined a particularly notable region of proximal 1q with a marked enrichment of genes which spans approximately 143–158 Mb corresponding to a 1q21-23 amplicon. The finding of high CNs of CKS1B and other genes in the 1q21~23 amplicon have been associated with disease progression and poor prognosis in MM. To investigate the possible mechanisms for focal gene amplification in this region, we identified 70 patients showing gain of 1q by G-banding. We then performed a comprehensive metaphase analysis utilizing fluorescence in situ hybridization (FISH) and spectral karyotyping to further characterize the karyotypic aberrations. Six FISH probes spanning the 1q12~23 region were used, including among others, probes for satII/III at 1q12 in the pericentromeric region to demark a proximal boundary, CKS1B at 1q21 to demark a point near the center of the amplicon, and RP11-57D16 at 1q25.2 to demark a distal boundary. In seven patients (10%) evidence for at least one breakage-fusion-bridge (BFB) cycle involving 1q12~23 in an inverted duplication was found. Strikingly, in three patients (4%) extended ladder-like structures of 1q12~23 inverted duplications were identified with up to 18 copies of CKS1B in contiguous duplicated regions. In these patients, the “amplicon ladders” showed the progression from two, to four, to eight copies, of CKS1B in different cells. Several key structures that are predicted intermediates in BFB cycles were observed in these patients, including equally spaced organization of amplicons, isodicentric chromosomes 1 with a clustering of breakpoints in the duplicated 1q12 pericentromeric regions, inverted repeat organization of amplicons along the same chromosome arm, and deletion of sequences distal to the amplified region. In these patients, site-specific breakage in the 1q12 pericentromeric heterochromatin mediated the organization of the BFB cycles by ultimately bracketing both the proximal and distal boundaries of the amplicon. Two chromosomal mechanisms have been described for the initiation of BFB cycles in tumor cells: the telomere fusion model in which chromosome breakage is induced by shortened or dysfunctional telomeres, and the alternative mechanism whereby replication stress and/or delay induces chromosome breakage in common fragile sites (CFS). The site-specific chromosome breakage in the satII/III sequences in the different phases of the intermediate chromosome structures identified here strongly support the concept that the BFB cycles were initiated by a CFS mechanism. A possible candidate fragile site in the 1q12 pericentromeric region is FRA1J, a known 5-azacytidine fragile site. It is well established that 5-azacytidine is a methyl transferase inhibitor which induces hypomethylation of the 1q12 pericentromeric DNA in metaphase chromosomes resulting in a characteristic pattern of pericentromeric decondensation and chromosome breakage. Our findings provide the first evidence for the BFB cycle mechanism of gene amplification in MM, and that the amplification process is induced by the secondary activation of a CFS in the 1q12 pericentromeric heterochromatin.