Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia
Proceedings of the National Academy of Sciences of the United States of America
Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the Western hemisphere. Tumor-specific chromosomal translocations, characteristic findings in several human malignancies that directly lead to malignant transformation, have not been identified in CLL. Using paired-end transcriptome sequencing, we identified recurrent and reciprocal RNA chimeras involving yippee like 5 (YPEL5) and serine/threonine-protein phosphatase PP1-beta-catalytic subunit (PPP1CB) in CLL.
... o of seven index cases (28%) harbored the reciprocal RNA chimeras in our initial screening. Using quantitative real-time PCR (q real-time PCR), YPEL5/PPP1CB and PPP1CB/YPEL5 fusion transcripts were detected in 97 of 103 CLL samples (95%) but not in paired normal samples, benign lymphocytes, or various unrelated cancers. Whole-genome sequencing and Southern blotting demonstrated no evidence for a genomic fusion between YPEL5 and PPP1CB. YPEL5/PPP1CB chimera, when introduced into mammalian cells, expressed a truncated PPP1CB protein that demonstrated diminished phosphatase activity. PPP1CB silencing resulted in enhanced proliferation and colony formation of MEC1 and JVM3 cells, implying a role in the pathogenesis of mature B-cell leukemia. These studies uncover a potential role for recurrent RNA chimeras involving phosphatases in the pathogenesis of a common form of leukemia. chimera splicing | next-generation sequencing | serine/threonine phosphatase PP1-beta catalytic subunit | B-lymphoid malignancy B -cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in adults in Western countries (1). The most common recurrent cytogenetic abnormality in CLL is a deletion involving the 13q14.3 locus, which occurs in 50% of cases and targets miR-16-1, miR-15a, and DLEU2 (2-4). Twenty percent of CLLs exhibit trisomy 12 (5). Other recurrent abnormalities in CLL include del 11q22-23 (ATM) and 17p13 (targeting p53) (6, 7). Of clinical relevance, IgV mutational status and zeta-chain associated protein kinase-70 kD (ZAP-70) expression have been associated with distinct prognostic categories of B-CLL (8-10). Recently, mutations in NOTCH1 (12.2%), MYD88 (2.9%), and XPO1 (2.4%) have been identified using next-generation sequencing (11, 12). The NOTCH1 mutations occur more frequently in cases with unmutated variable regions of the Ig heavy chain genes, whereas the MYD88 mutations occur more frequently in mutated cases (11). Although the role of genomic events is well established in the pathogenesis of cancers, the contribution of posttranscriptional RNA processing, which plays a fundamental role in control of protein expression, is less well understood. Alternative splicing can affect the translation, localization, or degradation of mRNA (13) and frequently results in the production of multiple and functionally distinct protein isoforms (14). Importantly, alternative splicing and expression of abnormal splicing chimeras may contribute to cancer pathogenesis and are associated with prognostic significance (15, 16) . For example, alternative splicing of CD44 has been associated with enhancement of metastatic potential (17). Similarly, the glycolytic enzyme pyruvate kinase M is known to undergo alternative splicing to yield a protein product (PKM2) that regulates cancer metabolism (18). Alternative splicing of the tyrosine kinase SYK has been shown to promote oncogenesis in ovarian cancer cells (19). Indeed, chimeric transcripts that exert oncogenic effects have been described. Expression of an RNA chimera fusing CCND1 and TROP2 (TACSTD2) transcripts has been demonstrated to result in immortalization and transformation of human epithelial cells (16) . Intriguingly, reciprocal RNA splicing chimeras that are recurrent in specific forms of cancer have not been described. However, recent studies using next-generation sequencing have identified a recurrent nonreciprocal chimera involving SLC45A3 and ELK4 in prostate cancer by a cis-splicing mechanism without DNA-level rearrangement (20) (21) (22) . In this study we discovered recurrent reciprocal chimeric transcripts between YPEL5 and PPP1CB genes in CLL using whole-transcriptome sequencing. Whole-genome sequencing and extensive Southern blotting analyses revealed the wild-type configuration at both YPEL5 and PPP1CB gene loci, indicating that the chimeras resulted from RNA splicing events rather than a chromosomal rearrangement. Evaluation of the presence of the chimeric fusion by quantitative real-time PCR (q real-time PCR) in diverse hematopoietic neoplasia, normal B-and T-cell subsets, and nonlymphoid malignancies revealed selective expression of the chimeras in CLL. The RNA fusion chimera resulted in a truncated PPP1CB protein product with reduced enzymatic activity. Reduced expression of PPP1CB protein further enhanced the oncogenic phenotype in MEC1 and JVM3 B-cell leukemia cells. These results suggest a role for RNA splicing chimeras in the pathogenesis of CLL.