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 neoplasia, 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 expression 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) expression 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 24, 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. 1. (A) Mate-pair read mapping depicts the occurrence of YPEL5/PPP1CB and PPP1CB/YPEL5 chimera in CLL index samples. (B) Q real-time PCR validation of fusion transcripts in index CLL samples. cDNAs from two index CLL samples (C41, D51) were analyzed by q real-time PCR using SYBR green and custom primers to detect YPEL5/PPP1CB and PPP1CB/YPEL5. The Ct values for the fusions were normalized against their respective GAPDH values. Error bars represent means ± SEM. The analyses were done in technical triplicates and in three independent experiments. (C) Recurrent expression of YPEL5/PPP1CB and PPP1CB/ YPEL5 in CLL. SYBR green-based q real-time PCR analysis was performed in seven independent CLL cases other than index samples for detection of YPEL5/ PPP1CB and PPP1CB/YPEL5 and compared with different lymphoma-derived cell lines and solid tumors, such as prostate (Pros), gastric (Gas), and melanoma (Mel). The Ct values for the fusions were normalized against their respective GAPDH values. The results are representation of three independent experiments. (D) Sequence traces obtained by Sanger sequencing of the PCR amplicons obtained using YPEL5 and PPP1CB primers designed to amplify across the chimeric fusion transcripts. Schematic presentation shows the presence of sequences of paired end reads from PETS in the amplicons.

3036 | www.pnas.org/cgi/doi/10.1073/pnas.1214326110 Velusamy et al. Downloaded by guest on September 24, 2021 Fig. S1A). This aberration leads to generation of a protein in SYBR green-based q real-time PCR to measure the levels of which the first 28 amino acid residues are lost from the wild-type PPP1CB wild-type transcripts. The results show that there was PPP1CB protein, leading to a 299-aa residue truncated protein expression of wild-type PPP1CB at comparable levels to controls composed of residue 29 to residue 327. The N-terminally truncated that were negative for chimeras (Fig. S3). protein retains an intact PP2Ac phosphatase domain (Fig. S1A). In the reciprocal PPP1CB/YPEL5 fusion, exon 1 of PPP1CB is Genomic Analysis of YPEL5/PPP1CB Fusion. We performed whole- juxtaposed to exon 3 of YPEL5, generating a fusion transcript genome mate-pair sequencing on DNA isolated from two of the that encodes the full-length wild-type YPEL5 protein without RNA chimera-positive index cases of CLL, to determine whether a coding contribution from PPP1CB (Fig. S1A). a genomic rearrangement was responsible for the YPEL5/PPP1CB and reciprocal chimeric transcripts. Although these studies revealed Validation of YPEL5/PPP1CB and Reciprocal Fusion in Independent common structural alterations, such as deletion of 13q (Fig. Cases of CLL. We designed independent conventional gel-based, S4B), extensive analysis revealed no evidence of a genomic basis SYBR Green I, and fusion-specific hydrolysis (TaqMan)-based for a juxtaposition of YPEL5 and PPP1CB or junctional sequences YPEL5/PPP1CB q real-time PCR assays targeting the and indicating a gene fusion between the two genes at the DNA level. PPP1CB/YPEL5 fusions. Using these assays, we investigated 103 The YPEL5, PPP1CB, and intervening loci showed a normal cases of CLL, as well as 5 benign lymph node hyperplasias and pattern of expected ∼5-kb mate-pair spacings, representative of purified lymphocyte subpopulations, germinal center B cells, fi the source libraries, with no regions of apparent copy number naïve B cells, memory B cells, and T cells puri ed from hyper- gain or loss (Fig. 3 and Fig. S4B). plastic tonsils. Additionally, we investigated a total of 135 primary We performed Southern blot hybridization using DNA iso- samples of a diverse spectrum of primary human cancers in- lated from two of the index cases that yielded the YPEL5/ cluding mantle cell lymphoma (n = 43), acute myelogenous PPP1CB n = n = fusion by paired-end transcriptome sequencing, to leukemia ( 17), chronic myelogenous leukemia ( 10), further investigate the origin of the YPEL5/PPP1CB fusion. follicular lymphoma (FL) (n = 6), precursor B-cell acute lym- Southern blot hybridization using a 0.5-kb-long probe targeting phoblastic leukemia (n = 5), precursor T-cell acute lymphoblastic intron 2 of YPEL5 and DNA derived from the index samples leukemia (n = 5), Burkitt lymphoma (n = 5), marginal zone lymphoma (n = 4), prostate carcinoma (n = 14), gastric carci- did not reveal any novel nongerm line bands in independent n = n = experiments with three different restriction enzymes (EcoR1, noma ( 13), and malignant melanoma ( 13). In addition, Xba1 Nco1 B A B a total of 12 cell lines, including mantle cell lymphoma (n = 1), ,and )(Fig.3 and Fig. S5 and ). Similarly, n = Southern blot hybridization of DNA of index samples with FL/diffuse large B-cell lymphoma ( 3), acute myeloid leuke- PPP1CB n = n = three different probes (0.5 kb) targeting intron 2 of MEDICAL SCIENCES mia, ( 5), mast cell leukemia ( 1), prolymphocytoid B-cell Spe1 Xcm1 chronic lymphocytic leukemia (n = 1), and epithelial cancer (n = using two different restriction enzymes ( and ) also did 1), were tested. Strikingly, only the primary CLL specimens not show any such recombinations, indicative of absence of PPP1CB showed PCR evidence for the reciprocal fusion (Fig. 2 A and B). genomic rearrangements involving the locus (Fig. S6). To establish that the YPEL5/PPP1CB and reciprocal chimeras These results are supportive of RNA splicing events as the basis were expressed preferentially in the tumor cells, we examined for the YPEL5/PPP1CB chimera detected in CLLs by PETS paired samples (n = 5) comparing CLL cells immunoaffinity- and RT-PCR. enriched using B cell-specific anti-CD19 conjugated beads to We also performed FISH on interphase cells from YPEL5/ nonmalignant granulocytes obtained by immunoaffinity enrich- PPP1CB-positive CLL samples using break-apart probes flanking ment with an anti-CD13/33 mixture. In all cases, only the B-cell the ends of YPEL5 and PPP1CB gene (Fig. 3D). Our results show fractions containing CLL tumor cells revealed YPEL5/PPP1CB that both PPP1CB and YPEL5 probes stayed in close proximity to and reciprocal chimeras by q real-time PCR, whereas the gran- each other (Fig. 3D, yellow arrows), indicating absence of either ulocyte-cell fractions were negative (Fig. 2C). Further, to know copy number changes (amplification/deletion) or chromosomal whether the reciprocal chimeras affect the expression of wild-type rearrangement that results in breaking and dislocation of PPP1CB in chimera-positive patient samples, we performed flanking probes.

Fig. 2. (A) Extent of YPEL5/PPP1CB and PPP1CB/ A YPEL5/PPP1CB PPP1CB/YPEL5 B YPEL5/PPP1CB PPP1CB/YPEL5 YPEL5 expression in CLL, benign hyperplasias, other lymphoid malignancies, and solid tumors. AML, 120 120000 2500 acute myeloid leukemia; B-ALL, B-cell acute lym- 100 100000 2000 80000 phoblastic leukemia; BH, benign hyperplasia-BH; BL, 80 1500 Burkitt lymphoma; CML, chronic myeloid leukemia; 60 60000 1000 FL, follicular lymphoma; GC, gastric cancer; MCL, 40 40000 500 ’ % Occurrence 20000 Target over GAPDH mantle cell lymphoma; ML, melanoma MZL, mar- 20 Target over GAPDH ginal zone lymphoma; PC, prostate cancer; T-ALL, 0 0 0 Naïve GCB C38 D51 NCEB HEK Water Naïve GCB C38 D51 NCEB HEK Water T-cell acute lymphoblastic leukemia. (B)TaqManq Bcell CLL CLL 293 Bcell CLL CLL 293 real-time PCR analyses for fusion genes in germinal center B cells and naïve, memory B cells vs. CLL. (C) YPEL5/ C D PPP1CB Somatic acquisition of YPEL5/PPP1CB and PPP1CB/ YPEL5/PPP1CB PPP1CB/YPEL5 PPP1CB 120000 60000 YPEL5 in CLL. TaqMan q real-time PCR validation 100000 50000 c 12 312 3 showing expression of RNA chimeras only in tumor V cells. Q real-time PCR analyses for YPEL5/PPP1CB 80000 40000 and PPP1CB/YPEL5 transcripts in immunoaffinity 60000 30000 Flag/PPP1CB enriched CLLs using B cell-specific anti-CD19 beads 40000 20000 Target over GAPDH (C41 to D14) and to constitutional matched gran- 20000 Target over10000 GAPDH ulocytes material obtained by immunoaffinity en- 0 0 richment with an anti-CD13/33 mixture (matched Actin normal; MNC41 to MN D14). The data presented represent the results of technical triplicates of three independent experiments. (D) Expression of PPP1CB full-length (PP-FL) and truncated protein (PP-Tr) in HEK 293 cells. PPP1CB full-length gene and YPEL5/PPP1CB fusion chimera cloned in a mammalian expression vector with a Flag tag plasmid DNA of three separate clones were transfected to HEK 293 cells. After 48 h, the protein lysates of the cells were checked for expression of proteins by Western blots using Flag mouse monoclonal antibody.

Velusamy et al. PNAS | February 19, 2013 | vol. 110 | no. 8 | 3037 Downloaded by guest on September 24, 2021 A B Functional Analysis of the YPEL5/PPP1CB Fusion Product. We expressed CLL recombinant full-length PPP1CB (wild-type, PP-FL) or mutant Placenta D-84 Leukocytes D-6 CL-F Normal node Lymph PPP1CB protein lacking N-terminal 28-aa (truncated, PP-Tr) ALK YPEL5 proteins in Escherichia coli and assessed their catalytic activity by 10 performing an in vitro phosphatase assay, to determine whether Ex 1 Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 8 Int 1 Int1 Int2 Int3 Int4 Int 5 6 5 truncation by the fusion causes functional activation or loss of 4 PP (500bp) 1 3.5 Kb PPP1CB. Our results indicate that the truncated PPP1CB (PP-Tr) 3 ECoR1 ECoR1 protein demonstrated significantly less phosphatase activity (P < XbaI 3.4kb XbaI 2 0.01), as depicted by reduction in the absorbance compared with 1.5 Nco1 4.2k b Nco1 A 1 PP-FL (Fig. 4 ). GST fusion proteins alone had very minimal 15.2 kb cross-effects on the activity of the proteins. These results confirm C 0.5 that N-terminal truncation caused by YPEL5/PPP1CB fusion Chr 2 D Chr. 2p23.2 decreases the phosphatase activity of PPP1CB enzyme. PPP1CB YPEL5 PPP1CB YPEL5 fi 5’ 3’5’ 3’ Given our demonstration of signi cantly diminished func- Expected 28,828,130 28,879,310 30,223,254 30,2396,903 YPEL5/ RP11 - 1079D1 RP11 - 984I21 RP11 - 104E4 RP11 - 136A10 tional activity of the truncated PPP1CB encoded by the PPP1CB Break Apart Probe YPEL5 Break Apart Probe PPP1CB Deletion fusion, we sought to investigate the biologic consequences of functional inactivation of PPP1CB. To this end, we knocked Insertion down the expression of PPP1CB using a lentivirus-based shRNA Inversion approach and evaluated its effects on cell proliferation in

Duplication B-lymphocytic leukemia cell lines MEC1 (22) and JVM3 (24), as well as in National Institutes of Health (NIH) 3T3 and Ba/F3 cell lines. Water-soluble tetrazolium-1 (WST-1) cell proliferation Fig. 3. (A) Restriction sites map depicting the sites targeted in intron 2 of assays demonstrated that in comparison with control (P < 0.01), YPEL5 by different restriction enzymes. Two enzymes, EcoR1 and Xba1, were knockdown of PPP1CB resulted in increased cell proliferation in predicted to generate 3.4-kb and 4.2-kb fragments, respectively. Enzyme NIH 3T3 and Ba/F3 cells (Fig. S7). Importantly, PPP1CB si- Nco1 was used to generate a large fragment (15.2 kb) that encompasses the lencing also resulted in significant increases (P < 0.01) in pro- entire YPEL5 gene to identify break points in case the fusion chimera is liferation of both B-lymphocytic leukemia cell lines MEC1 and generated by YPEL5 transcript variant 4. A genomic probe (P1) spanning the JVM3 (Fig. 4B). exon 1 and a part of intron 2 was used for Southern hybridization. (B) We performed soft agar colony assays using PPP1CB knock- Southern blotting analyses of CLL and benign hyperplasias (normal lymph down in the above-mentioned cell lines along with scramble node). The conventional germ line bands generated by respective enzymes C are indicated by arrows. DNA from CLL samples exhibited the germ line shRNA-expressing cells as control. As shown in Fig. 4 and Fig. S7, configuration indicating the absence of genomic breakpoints within intron 2 PPP1CB silencing resulted in increased colony formation com- resulting in YPEL5 and PPP1CB fusions. (C) Whole-genome mate-pair se- pared with control scramble shRNA-expressing cells (P < 0.01). quencing results. The figure shows the PPP1CB-YPEL5 region of chr2p for Overall, these functional studies suggest that impaired PPP1CB C41 (SI_3560) index sample. Image elements from top to bottom are (i) function promotes an oncogenic phenotype in mature B-cell a chromosome band ideogram with a red box indicating the region zoomed lymphocytic leukemia-derived cells. below, (ii) chromosome coordinates, (iii) a gene track, with selected genes labeled, (iv) an estimate of the copy number (CN) across the region, obtained Discussion by comparing local fragment coverage to all autosomes, (v) a track of the Whole-transcriptome sequencing studies have confirmed that ∼5-kb inward facing (Expected) mate-pair fragments mapping within the multiple transcripts are generated from most single genes and region, where bars indicate the span of the fragment, and (vi) tracks rep- that alternative splicing plays a significant role in the production resenting anomalously mapped pairs that were too big (Deletion), too of protein diversity (25, 26). Gene deregulation by recurrent and small (Insertion), in the colinear orientation (Inversion), or in an outward characteristic structural alterations of the genome resulting in facing orientation (Duplication). (D) Genomic organization and FISH vali- chimeric fusions is a common mechanism underlying cancer dation of PPP1CB and YPEL5 in CLL. Schematic diagrams showing the ge- – nomic location of PPP1CB (Left)andYPEL5 (Right)genesonchromosome pathogenesis (27 29), but the role of aberrant splicing events 2p23.2. The green and red rectangles with BAC clone identification num- resulting in chimeric transcripts that participate in tumorigenesis bers indicate the 5′ (green) and 3′ (red) clones used in the generation of and progression is largely unexplored. break-apart probes. FISH analysis on CLL samples revealed no rearrange- In this study we have identified high frequency (>95%) re- ment in either of the probes. Arrows (yellow) indicate colocalization of ciprocal YPEL5/PPP1CB and PPP1CB/YPEL5 chimeric fusion 5′ and 3′ probes, indicating normal signal pattern in all of the interphase transcripts in CLL. The YPEL5/PPP1CB and reciprocal fusions cells analyzed. were detected selectively in CLL cases and not in other cancers. Genomic analyses using whole-genome sequencing, Southern blot hybridization, and FISH in index and other fusion-positive We performed IgH variable region sequencing to determine samples did not reveal a genomic basis for the generation of the the mutational status of the Ig genes expressed in 25 of the cases chimeric transcripts. The presence of YPEL5/PPP1CB and re- of CLL investigated in this study. These studies revealed that ciprocal fusions in the neoplastic CLL cells and their absence in 52% of the cases were mutated, and 48% were unmutated. This purified granulocytes from the same (pair-matched) patients indicates a comparable frequency of mutated and unmutated indicates selective expression of these fusion transcripts in the YPEL5/PPP1CB IgV cases in YPEL5/PPP1CB-positive cells. neoplastic cells. The chimera is thus inferred to represent a splicing fusion between noncontiguous genes (1.34 Expression of Protein Products from the YPEL5/PPP1CB Fusion. The Mb apart) wherein six coding genes are skipped within the YPEL5/PPP1CB spliced fusion. One of these genes ALK (2p23) has 29 exons that architecture of the fusion predicts the genera- B tion of a truncated PPP1CB protein product of 31 kDa (Fig. are intact and not involved in the fusion chimera (Fig. S1 ). YPEL5 is a member of the highly conserved YPEL (Yippee S1A). To investigate whether the YPEL5/PPP1CB chimeric fi like) gene family that encodes putative zinc-binding proteins transcript produces a functional protein product, we ampli ed (30). The wild-type YPEL5 gene encodes a 121-aa protein whose the full-length fusion transcript from index samples and cloned functions is not yet fully understood but seems to be involved in them into a mammalian expression vector. Introduction of these the cell cycle progression (31). The partner gene PPP1CB en- plasmids into HEK 293 cells resulted in synthesis of a truncated codes the serine/threonine-protein phosphatase PP1-β-catalytic PPP1CB protein that was smaller in size compared with wild- subunit (PPP1CB) in humans (32). PPP1CB is a 328-aa enzyme type PPP1CB protein (Fig. 2D). that is one of the three catalytic subunits of protein phosphatase

3038 | www.pnas.org/cgi/doi/10.1073/pnas.1214326110 Velusamy et al. Downloaded by guest on September 24, 2021 A B Given the fact that phosphatases are critically involved in cellular growth control and potentially in the development of cancer (38), we performed further functional studies to investigate a previously undescribed role for PPP1CB in cancer pathogenesis. Our studies revealed that functional inactivation of PPP1CB in B-CLL–related cells results in enhanced proliferation and increased colony formation, thus supporting a role for PPP1CB deregulation in B-CLL pathogenesis. Transcription-induced RNA chimeras have been previously recognized and thought to occur in physiologically normal tissues, or less frequently in neoplastic tissues wherein they are hypothesized to confer tumorigenic signals via gain of function or loss of function effects (39–41). Additionally, intergenic splicing C events resulting in differential expression of chimeric transcripts in tumoral tissues compared with normal tissues have been observed, although the selection mechanisms for maintaining these splicing events and chimeras are poorly understood (39, 40, 42, 43). Interestingly, it has been recognized that these intergenic splicing events between heterologous genes may mark the breakpoint sites that are targeted by chromosomal translocations at the genomic level (42, 44). Indeed, intra- and interchromosomal chimeric fusions arising from cis or trans splicing events with Fig. 4. (A) Phosphatase activity of wild-type PPP1CB and truncated proteins. genes within intra- or interchromosomal loci have been de- GST-tagged PPP1CB full-length (GST-PP-FL) or truncated proteins (GST-PP-Tr) scribed and implicated in the pathogenesis of other neoplasia were expressed in BL21 E. coli, affinity purified, and used for malachite (16, 18, 19, 21, 42, 44). In the present study however, no evidence green-based serine/threonine phosphatase assay. GST protein alone served for breakpoints at the genomic level that account for the YPEL5/ as control. The absorbance at 650 nm is presented in graph form. (Middle) PPP1CB and reciprocal chimera was identified. Further, we did Markedly diminished phosphatase activity exhibited by the truncated protein. not observe a high number of sequencing reads in our whole- (Bottom) Equal amounts of proteins were used for the assay. The results are transcriptome sequencing experiments, raising the possibility of representative of three independent experiments. *Statistical significance at expression of fusion chimera in a minority of cells. MEDICAL SCIENCES the .01 level (two-sided t test). Error bars represent means ± SEM. (B) Func- Chimeric RNAs may occur in the absence of genomic re- tional consequences of PPP1CB down-regulation. (B, i and B, ii)Repre- combination through intermolecular splicing of pre-mRNA sentative Western blot showing the knockdown efficiency of PPP1CB protein transcripts from genes at different genomic loci. These may oc- expression in stable MEC1 (i) and JVM3 (ii) cells expressing either of scramble cur as a result of errors associated with the spliceosomal ma- and PPP1CB shRNA. (B, iii and B, iv) WST cell proliferation assays in MEC1 (iii) chinery (45). Recently two independent studies have reported and JVM3 (iv) cells. *Statistical significance at the .01 level (two-sided t test). Error bars represent means ± SEM. (C)Colony-formationcellassaysinMEC1 mutations affecting the spliceosomal U2 snRNRP subunit SF3B1 and JVM3 cells stably expressing control shRNA and PPP1CB depleting shRNA. in up to 10% of CLL cases (46, 47). Although such alterations may contribute to abnormal activity of the splicing machinery, The total number of colonies observed in each condition was enumerated YPEL5/PPP1CB after 14–16 d and presented as bar graphs. The experiments are conducted the high frequency of the and reciprocal chi- with technical triplicates and repeated twice. *Statistical significance at mera observed in the present study suggests a role for aberrant the .01 level (two-sided t test). Error bars represent means ± SEM. (Lower) splicing events in the genesis of this prevalent chimera in CLL. Representative soft agar plates showing enhanced colony-forming capacity of Nevertheless, although our Southern blotting and genomic se- MEC1 and JVM3 cells that express low levels of PPP1CB. quencing studies did not reveal evidence for a genomic mechanism for generation of the reciprocal chimera, these results do not completely exclude the possibility of complex genomic events 1 (PP1), which itself is a subunit of large holoenzyme complex. occurring in a small proportion of the tumor cells and generating The PP1 family of protein phosphatases is involved in regulation the RNA chimera. of a variety of cellular processes, including cell division (33), In conclusion, transcriptome-wide paired-end sequencing is HIV-1 viral transcription (34), glycogen metabolism, muscle advantageous for the discovery of chimeric transcripts involved contractility, protein synthesis (35), and RNA splicing (36). in cancer development that have defied identification by other The YPEL5/PPP1CB RNA chimera observed in the present methods. In particular, the identification of reciprocal RNA study results from the juxtaposition of 5′ untranslated sequences chimeric transcripts involving YPEL5 and PPP1CB with func- from exon 1 of YPEL5 to exon 2 of PPP1CB, predicted to result tional protein and biologic consequences but absent in pair- in a truncated PPP1CB (299-aa) protein rather than a full-length matched constitutional cells have important implications for wild-type protein containing 328 aa. This is further confirmed a role of RNA splicing chimeras in the pathogenesis of CLL. The by introduction of YPEL5/PPP1CB fusion chimera into HEK presence of this fusion in cases of CLL irrespective of IgH mu- 293 cells resulting in synthesis of truncated PPP1CB protein, tational status, trisomy 12, or deletion 13q status suggests a broad confirming the translation of chimeric transcripts into protein role in the pathogenesis of the disease. Given its high frequency products. Functional studies revealed that the truncated PPP1CB and specificity, it is anticipated that identification of this fusion protein exhibited significantly decreased phosphatase activity in will impact the molecular diagnosis and possibly the treatment of in vitro enzymatic assays. These results are consistent with studies CLL. Our discovery of the YPEL5/PPP1CB fusion in CLL also demonstrating that deletion of the N-terminal domains of highlights the potential participation of deregulated phospha- members of the PP1 family of protein phosphatases alters the tases in the pathogenesis of CLL. affinity of these enzymes for their substrates, indicating the regulatory role for N-terminal sequences on the substrate spec- Methods fi fi i city and catalytic ef ciency of the enzyme (37). Expression of Index Samples. Cryopreserved cell suspensions from a total of five cases with both wild-type PPP1CB and YPEL5/PPP1CB chimera in B-CLL characteristic clinical and immunophenotypic features of CLL were obtained samples indicates that the truncated PPP1CB protein expressed from the flow cytometry laboratories of the University of Utah/Associated as a consequence of the RNA chimeric fusion may function as Regional and University Pathologists (ARUP) and the University of Michigan. a dominant negative protein inhibiting the function of the wild- The research use of these residual specimens was approved at both institu- type protein. tions (Utah: IRB 11905; Michigan: IRB HUM00023256). The mean age of the

Velusamy et al. PNAS | February 19, 2013 | vol. 110 | no. 8 | 3039 Downloaded by guest on September 24, 2021 patients was 65 y (Table S2). The samples were selected on the basis of flow Genomic Southern Blotting and Molecular Cloning of YPEL5/PPP1CB and Truncated cytometry-based enumeration of tumor cells such that they contained >80% PPP1CB Protein Expression. Genomic Southern blotting (49), cloning of YPEL5/ of tumor cells. RNA isolation and cDNA library preparation for whole- PPP1CB, and expression of truncated PPP1CB protein expression was per- transcriptome sequencing were performed as previously described (48) formed according to standard procedures and is detailed SI Methods.FISH,Ig fi with minor modi cations. heavy chain variable region gene expression analysis, serine/threonine phosphatase assay, and cell proliferation and colony formation assay were per- Nomination of Candidate RNA Chimeras. Mate-pair transcriptome reads were formed as described in SI Methods. mapped to the human genome (hg18) and RefSeq transcripts, allowing up to two mismatches, with the Illumina Genome Analyzer Pipeline software ACKNOWLEDGMENTS. This study was supported by National Institutes of fi ELAND (Ef cient Alignment of Nucleotide Databases). Sequence alignments Health Grants CA136905 and DE019249 (to K.S.J.E.-J.) and CA129528 (to were subsequently processed to nominate gene fusions, using previously M.S.L.). A.M.C. is an A. Alfred Taubman Scholar and is supported by a grant described bioinformatics methodology briefly described in SI Methods (22). from the Doris Duke Charitable Foundation.

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