Letters to the Editor 1600 CONFLICT OF INTEREST 2 Sokol L, Loughran TP Jr. Large granular lymphocyte leukemia. Oncologist 2006; 11: WK, CH, TH and SS are part owners of the MLL Munich Leukemia Laboratory. AF and 263–273. VG are employed by the MLL Munich Leukemia Laboratory. 3 Koskela HL, Eldfors S, Ellonen P, van Adrichem AJ, Kuusanmaki H, Andersson EI et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med 2012; 366: 1905–1913. ACKNOWLEDGEMENTS 4 Jerez A, Clemente MJ, Makishima H, Koskela H, Leblanc F, Ng KP et al. STAT3 mutations unify the pathogenesis of chronic lymphoproliferative dis- We thank all clinicians for sending samples to our laboratory for diagnostic purposes, orders of NK cells and T cell large granular lymphocyte leukemia. Blood 2012; 120: and for providing clinical information and follow-up data. In addition, we would like 3048–3057. to thank all co-workers at the MLL Munich Leukemia Laboratory for approaching 5 Kern W, Bacher U, Haferlach C, Alpermann T, Dicker F, Schnittger S et al. together many aspects in the field of leukemia diagnostics and research. In addition, Frequency and prognostic impact of the aberrant CD8 expression in 5,523 we are grateful for the data management support performed by Tamara Alpermann. patients with chronic lymphocytic leukemia. Cytometry B Clin Cytom 2012; 82: 145–150. A Fasan, W Kern, V Grossmann, C Haferlach, 6 van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL T Haferlach and S Schnittger et al. Design and standardization of PCR primers and protocols for detection of MLL Munich Leukemia Laboratory, Munich, Germany clonal immunoglobulin and T-cell receptor gene recombinations in suspect lym- E-mail: [email protected] phoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257–2317. 7 Lamy T, Loughran TP Jr. How I treat LGL leukemia. Blood 2011; 117: 2764–2774. 8 Sosin MD, Handa SI. Spontaneous remission of large granular lymphocytic REFERENCES leukaemia. Int J Clin Pract 2003; 57: 551–552. 1 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. WHO Classifi- 9 Shichishima T, Kawaguchi M, Ono N, Oshimi K, Nakamura N, Maruyama Y. cation of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. (International Gammadelta T-cell large granular lymphocyte (LGL) leukemia with spontaneous Agency for Research on Cancer (IARC): Lyon, 2008. remission. Am J Hematol 2004; 75: 168–172.

Frequent somatic mutations in components of the RNA processing machinery in chronic lymphocytic leukemia

Leukemia (2013) 27, 1600–1603; doi:10.1038/leu.2012.344 contained four genes encoding factors, which has known roles in RNA splicing (SF3B1, SFRS1/SRSF1, U2AF65/U2AF2 and BRUNOL4/ CELF4).4 In addition, we found three genes encoding RNA-export- Chronic lymphocytic leukemia (CLL) is a common neoplasia of B machinery factors (NXF1, XPO1 and DDX3X).4 This prompted us to lymphocytes with a heterogeneous clinical course ranging from investigate the role of mutations affecting the RNA processing an indolent disorder, with a normal lifespan, to an aggressive machinery, not limited to splicing, in the development of CLL. 1 disease and short survival. The genetic mutations and associated To assess the involvement of RNA-processing factors in the molecular alterations that are able to accurately recapitulate the development of CLL, we extended our previous high-throughput 2–5 molecular pathogenesis of CLL are only now coming to light. sequencing experiment in 105 patients to a cohort of 140 patients. Foremost amongst the mutated genes is SF3B1, a core component Thus, we compared the whole exomes of tumor and normal of the RNA splicing machinery. SF3B1 mutations were found to be samples from each patient with the Sidro´n algorithm somatically acquired in B10% of CLL patients, and correlate with (Supplementary Methods). With this method, we identified 52 4–7 a more aggressive behavior of the disease. Consistently, the somatic mutations in 44 patients affecting 29 genes (Figure 1 and recent identification in myeloid neoplasms of recurrent mutations Supplementary Table 1). This suggests that mutations on these in SF3B1 and other spliceosome components in a mutually factors may contribute significantly to CLL development. Con- exclusive manner suggests a common functional consequence in sistent with this, only 3 out of the 43 missense mutations 8–10 the regulation of RNA splicing. identified were predicted as neutral for the function of the protein Virtually all of the eukaryotic mRNAs are synthesized as (Supplementary Table 1). We compared this frequency with the precursor molecules that need to be extensively processed in background proportion of neutral mutations, as estimated with order to serve as a blueprint for proteins. The most prevalent steps the same method on a set of randomly chosen mutations in genes are the capping reaction at the 50-end, the removal of intervening not related to RNA processing (Supplementary Methods). The sequences by splicing, formation of 30-end poly (A)-tails and the proportion of predicted neutral mutations in RNA-processing- nuclear export of the generated mature mRNA. Other RNA related genes was significantly lower than in the background (3/43 molecules undergo some of these maturation steps. Thus, versus 39/122, P ¼ 0.009). The remaining nine mutations are 50-end capping and transport to the cytosol are necessary to predicted to cause severe effects in six genes through frameshifts, produce functional small nuclear ribonucleoproteins (snRNP), premature stop codons or missed splicing sites. Notably, two of 11 which in turn catalyze mRNA splicing. Although these the mutated genes, EIF4A3 and MAGOH, belong to the exon maturation steps affect most transcripts in the cell, deregulation junction complex (EJC). These genes, together with RBM8A and of splicing has recently been recognized as a driver of oncogenic CASC3, form the core of this complex,14 which suggests that the 12 processes. Moreover, it has been shown that the anti-tumoral EJC core might constitute a third target for somatic mutations mechanism of the drug spliceostatin A involves altered fidelity of in CLL. splice-site recognition and changes in alternative splicing, In spite of this evidence, several of the studied genes show low 13 particularly of cell-cycle-control genes. With these antecedents, somatic-mutation frequencies, suggesting that most of the we observed that the list of the 60 genes with the highest encoded factors may not be driving tumoral progression. Also, corrected mutational frequencies in CLL (RM-CLL genes class 1) the complementation pattern of these mutations does not

Accepted article preview online 28 November 2012; advance online publication, 18 December 2012

Leukemia (2013) 1567 – 1614 & 2013 Macmillan Publishers Limited Letters to the Editor 1601

Figure 1. Somatically acquired mutations in components of the RNA processing pathway in CLL. Presented is an overview illustrating the individual components of the RNA-processing pathway, with those components identified as being somatically mutated highlighted (*) and the mutated protein listed in red. Initially, nascent pre-mRNA transcripts undergo 50 capping and binding of the cap-binding complex (CBC), followed by the formation of the major spliceosome, the machinery responsible for the removal of pre-mRNA introns via a stepwise mechanism. Initial assembly steps include formation of pre-spliceosome complex A (top left nuclear complex) involving recognition of the 50 splice site by U1 snRNP (an interaction stabilized by members of the serine-arginine-rich (SR) protein family) and recognition of the 30 SS region by the U2 Auxiliary factor U2AF and by U2snRNP. U2AF binds to the intronic polypyrimidine tract and 30SS, and facilitates binding of U2 snRNP to the branch-point sequence. Stable U2 snRNP binding requires stabilizing interactions of the U2 snRNP heteromeric protein complexes SF3A and SF3B with the pre-mRNA and/or the 65 kDa subunit of U2AF. Transition to spliceosome complex B (bottom left nuclear complex) involves recruitment of the pre-assembled U4/U6/U5 tri-SNP and more than 35 non-RNP proteins, including protein components of the PRP19 and retention and splicing (RES) complexes. Activation of the spliceosome, a requirement for the first catalytic step of splicing and conversion to complex C (bottom right complex), requires the release of U1 and U4 snRNPs, followed by the second catalytic splicing step and dissociation of the spliceosome, and the simultaneous binding of the exon-junction complex (EJC) a few nucleotides upstream of the exon junction. Processed mRNA is exported from the nucleus mainly via interactions with the non-karyopherin heterodimer of NXF1 and NXT1, although a subset of mRNAs are exported via the karyopherin XPO1 and its binding partner DDX3X. suggest a clear underlying biochemical mechanism (Figure 2). To advance in the characterization of somatic mutations Therefore, we decided to focus on U2 snRNP and RNA-transport in U2 snRNP and RNA-transport factors, we performed factors, whose genes feature frequent somatic mutations in a a gene set expression analysis on RNA-Seq experiments large number of CLL patients. As the U2 snRNP complex is from 12 tumor samples with U2 snRNP complex mutations involved in splicing of mRNA, we then studied the consequences compared to a control set of 42 tumor samples without mutations of somatic mutations affecting these factors in mRNA splicing and in known RNA-processing factors (Supplementary Table 2). expression patterns. In this analysis, we used AceView to classify Likewise, we compared six tumor samples with RNA the splicing sites predicted by RNA-Seq of 12 tumor samples with transport mutations with 11 IGHV-unmutated tumor samples mutations in U2 snRNP factors and 42 control tumor samples without mutations in known RNA-processing factors (Supplementary Table 2) as known (described in AceView) or (Supplementary Table 2). A direct comparison of the coverage of novel (otherwise), and calculated the coverage for each of those all transcripts in each sample identified those genes whose sites (Supplementary Methods). As previously reported for SF3B1, expression is most likely to be affected by mutations in the somatic mutation of RNA-processing factors does not affect those factors (Supplementary Table 3). To extract information on overall splicing accuracy as assessed by RNA-Seq experiments. the putative effects of those changes, we performed Thus, the ratio of reads covering the known splicing sites versus gene set enrichment analyses. These analyses identified several reads covering novel splicing sites is similar in U2 snRNP-mutated significant sets, whose genes are differentially expressed in tumors (9.42±0.24) and control samples (9.46±0.36). Therefore, one of the groups (Supplementary Table 4). Interestingly, the even if the effect of mutations in splicing factors is mediated by FRS2/PLC cascade is enriched in genes with differential splicing aberrations, these changes must be localized in a few expression in samples with somatic mutations in RNA-transport genes, as has been shown for FOXP1.4 This is not surprising, as factors. The FRS2/PLC cascade regulates multiple downstream splicing is required for the synthesis of most proteins and RNAs, processes, including Akt phosphorylation and phosphatidyl and widespread changes in this process are likely to result in cell inositol hydrolysis, some of which have been related to death. In addition, mutations in factors involved in RNA RNA export.15 Therefore, mutations in the RNA-export machinery metabolism may affect the ratio between RNA isoforms, their might act in concert with deregulated FRS2/PLC signaling to export to the cytoplasm or the decay of the mRNA, leading to facilitate the development of CLL. Further experiments will be changes in the expression of proteins important for tumor necessary to validate this hypothesis and ascertain the nature of progression. this putative link.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1567 – 1614 Letters to the Editor 1602 282 266 013 006 053 900 171 323 082 124 136 008 189 324 182 019 275 350 012 188 197 144 305 785 091 358 083 023 319 280 322 010 274 100 270 159 033 174 155 328 156 758 029 016 SF3B1 U2AF2 SRSF1 U2 snRNP SRSF7 RBMX ZRSR2 NXF1 XPO1 RNA transport DDX3X PRPF19 CDC5L Prp19 complex BUD13 TET1 CDC2L2 RNF113A Other-spliceosome RNPC3 SKIV2L2 MPHOSPH10

EIF4A3 Exon junction complex MAGOH NCBP2 Cap-binding complex MBNL2 RBFOX1 Alternative splicing CELF4 CARM1 CPEB3 mRNA decay DDX6 CSTF2 Polyadenylation TDRD9 piRNA processing

NOTCH1 MYD88 ATM POT1 SI IGHV-Unmutated Figure 2. Distribution of somatic mutations in genes involved in RNA processing in CLL patients. Each red box indicates the somatic mutation of a gene (row) in a tumor sample (column). Mutations in other frequently mutated genes and the mutational status of IGHV are shown at the bottom.

Finally, we investigated the clinical consequences of somatic and the RNA-transport machinery. However, we have provided mutations in RNA processing factors. To this end, we reviewed the evidence of clinical disparities between patients with mutations in clinical evolution of patients carrying somatic mutations in U2 SF3B1 and patients with mutations in other components of the U2 snRNP or RNA-transport factors compared to patients with no snRNP spliceosome. Finally, we have shown for the first time the mutations in each of those pathways (Supplementary Table 5). putative clinical relevance of somatic mutations in RNA-transport This analysis showed that somatic mutations in the RNA- factors. Therefore, the RNA-maturation pathway provides attrac- processing machinery are heterogeneous in clinical and biological tive targets for the development of specific drugs that may be terms. As previously reported,4–7 somatic mutations in SF3B1 are useful in the treatment of a significant percentage of CLL patients. correlated with unfavorable clinical and biological parameters. However, a multivariate Cox analysis showed that only SF3B1 mutation, and not mutation in other factors of the U2 snRNP CONFLICT OF INTEREST spliceosome, has independent prognostic value. This suggests The authors declare no conflict of interest. that, in spite of the biochemical relationship between these genes, somatic mutations affecting different factors of the U2 snRNP ACKNOWLEDGEMENTS spliceosome entail different clinical consequences. Therefore, the This work was funded by the Spanish Ministry of Science and Innovation (MICINN) first approach to pharmacological targeting of this complex in CLL through the Instituto de Salud Carlos III (ISCIII) and Red Tema´tica de Investigacio´ n del 13 should focus on drugs that bind to SF3B1, which might be useful Ca´ncer (RTICC) del ISCIII. CL-O is an Investigator of the Botı´n Foundation. We are against aggressive forms of the disease. On the other hand, grateful to all members of the CLL Spanish Consortium for their continuous support mutations affecting RNA-transport factors are associated with to this project. We are also very grateful to all patients with CLL who have biological features related to adverse prognosis, notably lack of participated in this study. somatic hypermutation (Supplementary Table 5). This result singles out RNA transport as a new factor in the development and AJ Ramsay1, D Rodrı´guez1, N Villamor2, A Kwarciak1, evolution of this aggressive form of CLL, and suggests that the JR Tejedor3, J Valca´rcel3,ALo´pez-Guillermo2, A Martı´nez-Trillos2, pharmacological targeting of this pathway might provide new drugs XS Puente1, E Campo2,CLo´pez-Otı´n1 and V Quesada1 with important clinical implications for the treatment of these 1Departamento de Bioquı´mica y Biologı´a Molecular, recalcitrant cases. Further studies will be necessary to gain statistical Instituto Universitario de Oncologı´a-IUOPA, power and to ascertain the putative mechanisms underlying this Universidad de Oviedo, Oviedo, Spain; relationship, which might prove valuable for the prediction of the 2Unidad de Hematopatologı´a, Servicio de Anatomı´a Patolo´gica, clinical outcome of CLL from early stages of the disease. Hospital Clı´nic, Universitat de Barcelona, IDIBAPS, Taken together, these data suggest that somatic mutations Barcelona, Spain and affecting some factors in the RNA processing pathway contribute 3Centre de Regulacio´ Geno`mica and Universitat Pompeu Fabra, to the development of CLL. Furthermore, the preferential targets Barcelona, Spain of these mutations are components of the U2 snRNP spliceosome E-mail: [email protected]

Leukemia (2013) 1567 – 1614 & 2013 Macmillan Publishers Limited Letters to the Editor 1603 REFERENCES 8 Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R et al. Frequent 1 Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med 1995; 333: pathway mutations of splicing machinery in myelodysplasia. Nature 2011; 478: 64–69. 1052–1057. 9 Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D et al. 2 Zenz T, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From pathogenesis to Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med treatment of chronic lymphocytic leukaemia. Nat Rev Cancer 2010; 10: 37–50. 2011; 365: 1384–1395. 3 Puente XS, Pinyol M, Quesada V, Conde L, Ordonez GR, Villamor N et al. Whole- 10 Damm F, Nguyen-Khac F, Fontenay M, Bernard OA. Spliceosome and other novel genome sequencing identifies recurrent mutations in chronic lymphocytic leu- mutations in chronic lymphocytic leukemia and myeloid malignancies. Leukemia kaemia. Nature 2011; 475: 101–105. 2012; 26: 2027–2031. 4 Quesada V, Conde L, Villamor N, Ordonez GR, Jares P, Bassaganyas L et al. Exome 11 Wahl MC, Will CL, Luhrmann R. The spliceosome: design principles of a dynamic sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in RNP machine. Cell 2009; 136: 701–718. chronic lymphocytic leukemia. Nat Genet 2012; 44: 47–52. 12 David CJ, Manley JL. Alternative pre-mRNA splicing regulation in cancer: pathways 5 Wang L, Lawrence MS, Wan Y, Stojanov P, Sougnez C, Stevenson K et al. SF3B1 and programs unhinged. Genes Dev 2010; 24: 2343–2364. and other novel cancer genes in chronic lymphocytic leukemia. N Engl J Med 13 Corrionero A, Minana B, Valcarcel J. Reduced fidelity of branch point recognition 2011; 365: 2497–2506. and alternative splicing induced by the anti-tumor drug spliceostatin A. Genes Dev 6 Rossi D, Bruscaggin A, Spina V, Rasi S, Khiabanian H, Messina M et al. Mutations of 2011; 25: 445–459. the SF3B1 splicing factor in chronic lymphocytic leukemia: association with pro- 14 Le Hir H, Andersen GR. Structural insights into the exon junction complex. Curr gression and fludarabine-refractoriness. Blood 2011; 118: 6904–6908. Opin Struct Biol 2008; 18: 112–119. 7 Quesada V, Ramsay AJ, Lopez-Otin C. Chronic lymphocytic leukemia with SF3B1 15 Okada M, Ye K. Nuclear phosphoinositide signaling regulates messenger RNA mutation. N Engl J Med 2012; 366: 2530. export. RNA Biol 2009; 6: 12–16.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Novel TAL1 targets beyond protein-coding genes: identification of TAL1-regulated microRNAs in T-cell acute lymphoblastic leukemia

Leukemia (2013) 27, 1603–1606; doi:10.1038/leu.2013.63 being downregulated (Figures 1b–f). The three remaining micro- RNAs were excluded from subsequent analyses, as we stringently considered only those genes to be validated whose expression The basic helix-loop-helix transcription factor TAL1 is aberrantly was regulated in the predicted manner upon both TAL1 expressed in a majority of T-cell acute lymphoblastic leukemia overexpression and silencing (data not shown). (T-ALL) cases characterized by arrested development in the thymic Next, we evaluated whether the validated microRNAs were late cortical stage.1,2 Although TAL1 is a bona fide T-cell direct targets of TAL1 in T-ALL cells. For this purpose, we oncogene,3 with known direct targets in T-ALL,4 the aberrant scrutinized publicly available TAL1 ChIP-seq data (GEO accession transcriptional circuitry responsible for thymocyte transformation number GSE29181) for two T-ALL cell lines (JURKAT and is not yet fully understood. MicroRNAs are small, non-coding RNAs CCRF-CEM) and two primary T-ALL samples4 for the presence of that function as endogenous post-transcriptional repressors of TAL1-binding peaks up to 10 kb upstream of the transcription start protein-coding genes by binding to target sites in the 30-UTR of site of each microRNA gene. We identified one peak in a putative messenger RNAs.5 Aberrant expression of these molecules has promoter region for miR-146b (Supplementary Figure 1a), sug- been reported in several hematological malignancies, and gesting that this gene may be a transcriptional target of TAL1. microRNA expression signatures delineate ALL subgroups6 and Furthermore, two peaks were observed upstream of miR-223 can be interpreted in light of their variation during transcription start site (Supplementary Figure 1b). To confirm hematopoiesis.7 Individual microRNAs and networks have been these findings, we performed TAL1 ChIP-quantitative PCR in implicated in T-ALL,8 but the mechanisms responsible for altered JURKAT and CCRF-CEM cells using primers designed for the microRNA expression in this malignancy remain poorly explored. genomic areas covered by the two peaks in the miR-223 locus. We Here, we report the identification of novel, non-protein-coding confirmed that there is more than twofold enrichment, as TAL1 target genes, implicating microRNA genes as part of the compared with a mock ChIP performed against fibrillarin, in the transcriptional network downstream of TAL1 that may be amplified area within 3.5 kb upstream of the miR-223 transcription putatively involved in its oncogenic properties. start site (Figure 1g). These results indicate that miR-223 is a direct To identify a TAL1-dependent microRNA gene expression target of TAL1 in T-ALL. Interestingly, TAL1 appears to bind to a profile, we ectopically expressed TAL1 in the TAL1-negative previously described region containing a conserved proximal T-ALL cell line P12 and performed low-density array analysis (see genomic element with possible binding sites for the transcription Supplementary Data online for materials and methods). From 204 factor C/EBP.9 We did not find evidence from the available TAL1 detected microRNAs (out of 372 analyzed), we identified eight ChIP-seq data for direct binding of TAL1 to the remaining microRNA whose expression changed significantly upon TAL1 overexpres- genes, suggesting that miR-135a, miR-330-3p and miR-545 are sion (Figure 1a and Supplementary Tables 1 and 2). Subsequent indirectly regulated by TAL1, at least in the T-ALL cells analyzed. validation was performed by quantitative PCR analysis of each Interestingly, analysis of microRNA gene expression profiles in microRNA after enforcing or silencing the expression of TAL1. This different T-ALL subsets8 revealed that TAL/LMO primary samples allowed us to confirm the expected TAL1-mediated regulation for (integrating Sil-Tal1 þ and LMO þ cases, which frequently express five microRNAs: namely, miR-135a, miR-223 and miR-330-3p as high TAL1 levels) display higher levels of miR-223 (P ¼ 0.035) and being upregulated by TAL1; and miR-146b-5p and miR-545 as tend to express lower levels of miR-146b-5p (P ¼ 0.092) than other

Accepted article preview online 1 March 2013; advance online publication, 26 March 2013

& 2013 Macmillan Publishers Limited Leukemia (2013) 1567 – 1614