Identification of the Novel Chimeric Gene, PVT1-WWOX, in Multiple Myeloma with 8q24 Abnormality

Abstract 1823

Genetic abnormalities play a crucial role in the pathogenesis of various malignancies, including multiple myeloma (MM). Secondary cytogenetic abnormalities implicated in MM progression include 8q24 rearrangements, gain of the long arm of chromosome 1 (1q+), and loss of the short arm of chromosome 17 (17p-). The 8q24 rearrangements, including MYC and PVT1, have been identified by conventional cytogenetic analysis in 3.5–5.0% of MM patients and by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) in 9.5–20%. 8q24 rearrangements are frequently associated with advanced disease in MM patients and MM cell lines. Ig chromosomal translocations, such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% of MMs with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14 and 16q22, in which no candidate genes have been delineated so far, have also been identified as translocation partners. MYC has long been a possible candidate target gene for 8q24 rearrangements; however, many of the breakpoints within 8q24 have been assigned to various regions that encompassed more than 2 Mb centromeric or telomeric to MYC.

We have previously found frequent PVT1 rearrangements in MM. PVT1 rearrangements were detected in 7 of 12 patients (58.3%) and in 5 of 8 cell lines (62.5%) with 8q24 abnormalities. A combination of SKY, FISH, and oligonucleotide array identified several partner loci of PVT1 rearrangements, such as 4p16, 4q13, 13q13, 14q32 and 16q23–24, and identified a chimeric gene, PVT1 - NBEA, resulting in high expression of abnormal NBEA in a cell line with t(8;13)(q24;q13), AMU-MM1, suggesting PVT1 rearrangements play significant roles in myelomagenesis.

In this study, we analyzed RPMI8226 cell line in detail to identify other partner genes of PVT1 in these partner loci. SKY analysis revealed a complex karyotype including der(16)t(16;22)ins(16;8)(q23;q24) in this cell line. Oligonucleotide array analysis clearly demonstrated that the copy number change at 8q24 occurred within intron 1 of PVT1, and at 16q23, the copy number change occurred within intron 8 of WWOX, indicating that the translocation breakpoints of 8q24 and 16q23 were within intron 1 of PVT1 and intron 8 of WWOX, respectively. Based on these results, RT-PCR analysis was performed to detect chimeric products and direct sequencing of this product revealed the fusion of 5'-PVT1 exon 1 with WWOX exon 9-3'. The expression level of WWOX exon 9 was higher than WWOX exon 8–9 detected by semi-quantitative RT-PCR in RPMI8226, suggesting that high expression of WWOX derived from PVT1 - WWOX chimeric transcript, like PVT1 - NBEA.

WWOX is generally considered to be a candidate tumor suppressor gene, and known to have a proapoptotic effect by participating in the tumor necrosis factor (TNF) apoptotic pathway and via direct physical interaction with p53 and its homolog p73. However, immunohistochemistry revealed that WWOX protein level were rather elevated in gastric and breast carcinoma. Therefore, WWOX seemed not to act as tumor suppressor gene simply. Although both NBEA and WWOX are located at common fragile site, usually contributing to gene inactivation, FRA13A and FRA16D respectively, these genes highly express via fusion to PVT1. These findings indicate that PVT1 rearrangements play significant roles in myelomagenesis via translocation and fusion to cancer-related genes.