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Recurrent Reciprocal RNA Chimera Involving YPEL5 and PPP1CB in Chronic Lymphocytic Leukemia

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Recurrent Reciprocal RNA Chimera Involving YPEL5 and PPP1CB in Chronic Lymphocytic Leukemia Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia Thirunavukkarasu Velusamya,1, Nallasivam Palanisamya,b,c,1, Shanker Kalyana-Sundaramb,d, Anagh Anant Sahasrabuddhea, Christopher A. Mahere, Daniel R. Robinsonb, David W. Bahlerf, Timothy T. Cornellg, Thomas E. Wilsona,h, Megan S. Lima, Arul M. Chinnaiyana,b,c,i,2, and Kojo S. J. Elenitoba-Johnsona,2 Departments of aPathology, gPediatrics and Communicable Diseases, and hHuman Genetics, University of Michigan Medical School, Ann Arbor, MI 48109; bMichigan Center for Translational Pathology and cComprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109; dBharathidasan University, Thiruchirapalli 620024, India; eGenome Institute at Washington University in St. Louis, St. Louis, MO 63108; fDepartment of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112; and iHoward Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109 Edited by Carlo M. Croce, Ohio State University, Columbus, OH, and approved January 4, 2013 (received for review August 18, 2012) Chronic lymphocytic leukemia (CLL) is the most common form of of metastatic potential (17). Similarly, the glycolytic enzyme py- leukemia in adults in the Western hemisphere. Tumor-specific ruvate kinase M is known to undergo alternative splicing to yield chromosomal translocations, characteristic findings in several a protein product (PKM2) that regulates cancer metabolism human malignancies that directly lead to malignant transformation, (18). Alternative splicing of the tyrosine kinase SYK has been have not been identified in CLL. Using paired-end transcriptome shown to promote oncogenesis in ovarian cancer cells (19). In- sequencing, we identified recurrent and reciprocal RNA chimeras deed, chimeric transcripts that exert oncogenic effects have been involving yippee like 5 (YPEL5) and serine/threonine-protein described. Expression of an RNA chimera fusing CCND1 and phosphatase PP1-beta-catalytic subunit (PPP1CB)inCLL.Twoof TROP2 (TACSTD2) transcripts has been demonstrated to result in seven index cases (28%) harbored the reciprocal RNA chimeras in immortalization and transformation of human epithelial cells (16). our initial screening. Using quantitative real-time PCR (q real-time Intriguingly, reciprocal RNA splicing chimeras that are re- fi PCR), YPEL5/PPP1CB and PPP1CB/YPEL5 fusion transcripts were current in speci c forms of cancer have not been described. detected in 97 of 103 CLL samples (95%) but not in paired normal However, recent studies using next-generation sequencing have fi SLC45A3 samples, benign lymphocytes, or various unrelated cancers. identi ed a recurrent nonreciprocal chimera involving ELK4 cis MEDICAL SCIENCES Whole-genome sequencing and Southern blotting demonstrated and in prostate cancer by a -splicing mechanism without – no evidence for a genomic fusion between YPEL5 and PPP1CB. DNA-level rearrangement (20 22). In this study we discovered YPEL5 YPEL5/PPP1CB chimera, when introduced into mammalian cells, recurrent reciprocal chimeric transcripts between and PPP1CB expressed a truncated PPP1CB protein that demonstrated diminished genes in CLL using whole-transcriptome sequencing. phosphatase activity. PPP1CB silencing resulted in enhanced prolif- Whole-genome sequencing and extensive Southern blotting analyses revealed the wild-type configuration at both YPEL5 and eration and colony formation of MEC1 and JVM3 cells, implying PPP1CB a role in the pathogenesis of mature B-cell leukemia. These studies gene loci, indicating that the chimeras resulted from uncover a potential role for recurrent RNA chimeras involving phos- RNA splicing events rather than a chromosomal rearrangement. phatases in the pathogenesis of a common form of leukemia. Evaluation of the presence of the chimeric fusion by quantitative real-time PCR (q real-time PCR) in diverse hematopoietic neo- plasia, normal B- and T-cell subsets, and nonlymphoid malig- chimera splicing | next-generation sequencing | nancies revealed selective expression of the chimeras in CLL. serine/threonine phosphatase PP1-beta catalytic subunit | The RNA fusion chimera resulted in a truncated PPP1CB B-lymphoid malignancy protein product with reduced enzymatic activity. Reduced ex- pression of PPP1CB protein further enhanced the oncogenic -cell chronic lymphocytic leukemia (B-CLL) is the most com- phenotype in MEC1 and JVM3 B-cell leukemia cells. These Bmon form of leukemia in adults in Western countries (1). results suggest a role for RNA splicing chimeras in the patho- The most common recurrent cytogenetic abnormality in CLL is genesis of CLL. 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 Results percent of CLLs exhibit trisomy 12 (5). Other recurrent abnor- Chimera Candidates for CLL. Using our previously described analysis malities in CLL include del 11q22-23 (ATM) and 17p13 (tar- pipeline for chimera discovery (22, 23), we identified a total of geting p53) (6, 7). Of clinical relevance, IgV mutational status nine RNA chimeras in seven cases of CLL (Table S1). Of these and zeta-chain associated protein kinase-70 kD (ZAP-70) ex- pression have been associated with distinct prognostic categories candidates, six chimeras represented read-throughs of adjacent of B-CLL (8–10). Recently, mutations in NOTCH1 (12.2%), genes, two represented chimeras resulting from juxtaposition MYD88 (2.9%), and XPO1 (2.4%) have been identified using of transcripts encoded by genes on different chromosomes, and next-generation sequencing (11, 12). The NOTCH1 mutations oc- one represented chimeric transcripts from noncontiguous genes cur more frequently in cases with unmutated variable regions of the within the same chromosome (Table S1). The chimera repre- Ig heavy chain genes, whereas the MYD88 mutations occur more senting fusion of two discontinuous gene transcripts was a re- frequently in mutated cases (11). Although the role of genomic events is well established in the pathogenesis of cancers, the contribution of posttranscriptional Author contributions: N.P., M.S.L., A.M.C., and K.S.J.E.-J. designed research; T.V. and D.R.R. RNA processing, which plays a fundamental role in control of performed research; A.A.S., D.W.B., and T.T.C. contributed new reagents/analytic tools; T.V., protein expression, is less well understood. N.P., S.K.-S., C.A.M., and T.E.W. analyzed data; and T.V. and K.S.J.E.-J. wrote the paper. Alternative splicing can affect the translation, localization, or The authors declare no conflict of interest. degradation of mRNA (13) and frequently results in the pro- This article is a PNAS Direct Submission. duction of multiple and functionally distinct protein isoforms 1T.V. and N.P. contributed equally to this work. (14). Importantly, alternative splicing and expression of abnor- 2To whom correspondence may be addressed. E-mail: [email protected] or kojoelen@med. mal splicing chimeras may contribute to cancer pathogenesis and umich.edu. are associated with prognostic significance (15, 16). For example, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. alternative splicing of CD44 has been associated with enhancement 1073/pnas.1214326110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1214326110 PNAS | February 19, 2013 | vol. 110 | no. 8 | 3035–3040 Downloaded by guest on September 29, 2021 ciprocal chimeric fusion between YPEL5 and PPP1CB genes quencing of the PCR products from two independent PCR (average read count n = 3). reactions (115bp and 325 bp) (Fig. S1C) with different primer We prioritized the fusion between the YPEL5 and PPP1CB sets confirmed juxtaposition of sequences derived from YPEL5 genes for further analysis and validation based on its reciprocal and PPP1CB in both YPEL5/PPP1CB and the reciprocal PPP1CB/ nature, its recurrence (2/7), and exclusive identification in the CLLs YPEL5 (Fig. 1D)configurations in all six cases of CLL. compared with more than 500 other tumors within our compen- Analysis of the expression of YPEL5 and PPP1CB within our dium of tumors investigated by paired-end whole-transcriptome compendium of RNA seq data generated from >500 independent sequencing (PETS). Accordingly, we performed q real-time PCR samples representing different types of cancer revealed signifi- using cDNA prepared from the index clinical specimens of CLL cantly higher levels of YPEL5 expression in the CLLs, suggesting and confirmed the results of PETS in the index cases (Fig. 1 B a lineage or tissue-specific promoter activation, whereas PPP1CB and C). levels observed in CLL were comparable to those observed To characterize the complete YPEL5/PPP1CB and reciprocal across all other tumor samples and cell lines (Fig. S2). fusion transcripts, we performed Sanger sequencing of RT-PCR In the YPEL5/PPP1CB fusion, the noncoding exon 1 of YPEL5 products obtained from cDNA prepared from the two index is juxtaposed to exon 2 of PPP1CB. This juxtaposition results in (discovery) CLL samples and an additional eight cases of clinically loss of exon 1 of PPP1CB (containing the initiation codon) and and phenotypically typical CLL in which the YPEL5/PPP1CB utilization of an alternative initiation codon from exon 2, whereas fusion was detected by q real-time PCR. Direct Sanger se- YPEL5 contributes only 5′ untranslated sequences (Fig. 1A and A C (i) YPEL5/PPP1CB 8000 7000 6000 5000 Paired-end reads Paired-end reads 4000 G CCGCTCAGG TACGAGGATG CTGCTGGAGG AGATCTGTTG 3000 2000 1 179 4569 1 417 2855 1000 1 273 456 1 32 45 0 Target over GAPDH Target YPEL5-PPP1CB PPP1CB-YPEL5 +ve CLL test 1 417 4986 1 179 2617 Cell lines Pros Mel Gas 1732 456 321 45 PPP1CB/YPEL5 PPP1CB YPEL5 (ii) (NM_002709) (NM_001127401) 9000 8000 7000 6000 5000 Chr.2p23 4000 3000 2000 1000 Target over GAPDH Target 0 B 100000 YPEL5/PPP1CB PPP1CB/YPEL5 90000 +ve CLL test Cell lines Pros Mel Gas 80000 70000 G CCGCTCAGG TACGAGGATG CTGCTGGAGG GTTTTTAGAAC D 1 179 4569 1 417 2855 60000 1 273 456 1 32 45 50000 YPEL5-PPP1CB PPP1CB-YPEL5 40000 YPEL5 PPP1CB PPP1CB YPEL5 30000 Target over GAPDH Target 20000 10000 0 C41 YP-PP D51 YP-PP C41 PP-YP D51 PP-YP Fig.
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