Ambrogio, Lauren

Auclair, Daniel

Chmielecki, Juliann

Crago, Aimee M.

Frank, David A.

Heinrich, Michael C.

McKenna, Aaron

Meyerson, Matthew

O'Connor, Rachael

Rosenberg, Mara

Walker, Sarah R.

Nature Genetics

Harvard University - DASH 

 Whole exome sequencing identifies a recurrent NAB2-STAT6fusion in solitary fibrous tumors  (Article begins on next page)The Harvard community has made this article openly available.Please share how this access benefits you. Your story matters.Citation Chmielecki, J., A. M. Crago, M. Rosenberg, R. O'Connor, S. R.Walker, L. Ambrogio, D. Auclair, et al. 2013. “Whole exomesequencing identifies a recurrent NAB2-STAT6 fusion in solitaryfibrous tumors.” Nature genetics 45 (2): 131-132.doi:10.1038/ng.2522. http://dx.doi.org/10.1038/ng.2522.Published Version doi:10.1038/ng.2522Accessed February 19, 2015 3:57:24 PM ESTCitable Link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12152937Terms of Use This article was downloaded from Harvard University's DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth athttp://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAAWhole exome sequencing identifies a recurrent NAB2-STAT6fusion in solitary fibrous tumorsJuliann Chmielecki1,2,*, Aimee M. Crago3,*, Mara Rosenberg2, Rachael O'Connor3, Sarah R.Walker1,4,5, Lauren Ambrogio2, Daniel Auclair2, Aaron McKenna2, Michael C. Heinrich7,David A. Frank1,4,5, and Matthew Meyerson1,2,6,81Department of Medical Oncology, Dana-Farber Cancer Institute, Boston MA 02215 USA2Cancer Program, Broad Institute of Harvard and MIT, Cambridge MA 02143 USA3Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan-Kettering Cancer Center,New York NY 10065 USA4Department of Medicine, Brigham and Women's Hospital, Boston, MA 022155Department of Medicine, Harvard Medical School, Boston, MA 022156Department of Pathology, Harvard Medical School, Boston, MA 022157Division of Hematology and Medical Oncology, Portland VA Medical Center and OHSU KnightCancer Institute, Portland OR 97239 USASolitary fibrous tumors (SFTs) are rare mesenchymal tumors. Here, we describe theidentification of a NAB2-STAT6 fusion from whole exome sequencing of 17 SFTs. Analysisin 53 tumors confirmed the presence of seven variants of this fusion transcript in 29 tumors(55%), a lower bound for fusion frequency at this locus, suggesting that the NAB2-STAT6fusion is a distinct molecular feature of SFTs.To better understand the molecular lesions contributing to SFT, we performed whole exomesequencing of DNA isolated from SFTs and matched blood from 17 patients(Supplementary Table 1). On average, we sequenced a median of 28.9 Mb per tumor, and87.5% of the captured exons were covered to a depth of 20× or greater. An average of 22.5non-synonymous somatic mutations per tumor were observed across the 17 samples(median: 19; range: 12-41; median rate of 0.66 mutations/Mb; Supplementary Fig. 1). Thiscorresponded to 390 somatically mutated genes, of which one gene mutated in two samples(RBPJ) reached nominal statistical significance for recurrence (q < 0.1; SupplementaryTables 2 and 3; Supplementary Methods). Read count analysis of the tumor copy numberUsers may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research,subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms8To whom correspondence should be addressed: matthew_meyerson@dfci.harvard.edu.*These authors contributed equally to this work.Accession codes: Sequence data used for this analysis are available in dbGaP under accession number phs000568.v1.p1, and areavailable at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000568.v1.p1.Contributions: JC and MM conceived the project. JC, AMC, and MM wrote the manuscript with input from all co-authors. JC, AMC,and RO performed experiments. JC and MR performed computational analyses. AM contributed an unpublished algorithm to theanalysis. JC, AMC, MR, SRW, RO, DF, and MM analyzed the results. AMC and MCH contributed samples. LA and DA facilitatedtransfer, sequencing, and analysis of samples.Conflicts of interest: MM is a paid consultant for and equity holder in Foundation Medicine, a genomics-based oncology diagnosticscompany, and is a paid consultant for Novartis. MCH is a paid consultant for and equity holder in Molecular MD, a moleculardiagnostics company, and is a paid consultant for Novartis.NIH Public AccessAuthor ManuscriptNat Genet. Author manuscript; available in PMC 2014 April 11.Published in final edited form as:Nat Genet. 2013 February ; 45(2): 131–132. doi:10.1038/ng.2522.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptprofiles showed 11 tumors with structurally undisrupted genomes and no obvious gains orlosses, while six tumors showed a broad loss of chromosome 13, including two samples witha concurrent broad gain of chromosome 8 (Supplementary Fig. 2).Given that many soft tissue tumors are driven by genomic translocations,1we explored thepossibility that gene fusions may be contributing to tumorigenesis in SFT. Using wholeexome sequencing data, our fusion detection was limited to breaks that occurred withinexons or near intronexon boundaries. However, we identified 19 potential fusion events in10/17 tumor samples using algorithms designed for this application (Supplementary Table 4;Supplementary Methods).Rearrangements in 7/17 tumors represented in-frame fusions of the NAB2 and STAT6 genesboth located on chromosome 12 (Supplementary Fig. 3a). Sequence review identifiedpaired-end reads where one read mapped to an exonic region of NAB2 and its pair matemapped 1.8 to 5.2 kilobases before the transcription start site of STAT6 (Supplementary Fig.3b; Supplementary Table 4). Given that sequence mate pairs representing the two ends of aDNA fragment were mapped in opposite orientations on the same strand (SupplementaryFig. 3b), we concluded that this fusion was likely due to an intra-chromosomal inversionthat juxtaposed NAB2 and STAT6 (Fig. 1a). Normally, NAB2 and STAT6 are in oppositeorientations but the predicted inversions bring the two genes in close proximity in the sameorientation (Fig. 1a). In all cases, we also identified at least one sequence that spanned theNAB2/STAT6 fusion boundary. We confirmed the genomic breakpoint in two tumors bysequencing of PCR products generated with breakpoint-spanning primers (SupplementaryFig. 4). The fusion was not observed in DNA from matched normal tissue (data not shown).To analyze the expression and frequency of NAB2-STAT6 fusions, we performed RT-PCRusing primers placed within exons 2-3 of NAB2 and within exon 1 of STAT6, and cDNAgenerated from 53 tumor samples representing 48 patients (Supplementary Table 1). An RT-PCR product was identified in 27 tumors (51%) from 23 patients. Sequencing of theseproducts revealed multiple isoforms of the fusion that varied in the location of the fusionwithin NAB2 (Fig. 1b, Supplementary Fig. 4). The NAB2 sequence was followed by asequence belonging to the 5′ untranslated region of STAT6. Comparing the cDNA structureto the inversion structure identified at the genomic level indicates that the region betweenexon 5 of NAB2 and the beginning of STAT6 (∼10kb) is spliced during transcription or thata large portion of this intronic region is deleted when the chromosome is rearranged.Additionally, we identified rare NAB2-STAT6 fusions that occurred in downstream exons ofSTAT6 in 2/17 cases from whole exome sequencing data (see Supplementary Table 4).NAB2 encodes a transcriptional repressor of the zinc finger transcription factors EGR1 andEGR2, effectors of TGFβ signaling in smooth muscle.2,3 NAB2 contains two conserveddomains that interact with EGR1 to mediate multimerization and repress transcription,respectively.2,3 Functional alterations of NAB2 have not been demonstrated in cancer butloss is reported in prostate cancer, lung cancer, non-Hodgkin's lymphoma, andneuroblastoma, and overexpression in melanoma, Ewing sarcoma, andrhabdomyosarcoma.4-6STAT6 is a transcription factor that modulates signaling by IL-4 and IL-13 in the immunesystem.7 Activation of STAT family members in cancer underlies the hypothesis that theseproteins are potential therapeutic targets.8 STAT6 in particular has been associated withincreased proliferation and invasiveness in glioblastoma.9 Based on the properties of NAB2and the oncogenic potential of STAT6, we hypothesize that the NAB2-STAT6 fusiondimerizes through the oligomerization domain of NAB2, translocates to the nucleus, andChmielecki et al. Page 2Nat Genet. Author manuscript; available in PMC 2014 April 11.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptmodulates STAT6-dependent gene expression. In experimental systems, fusion of STAT3 toa portion of the estrogen receptor results in a similar phenomenon.10In summary, we have identified a novel NAB2-STAT6 fusion in at least half of SFT tumorsfrom whole exome sequencing data. Although most fusions are identified from wholegenome or whole transcriptome sequencing, this finding validates the use of exome data forthe discovery of fusions that occur mid-exon. The NAB2-STAT6 fusion appears to be uniqueto SFT samples as fusion analysis of approximately 713 unique tumor-normal pairs from 5tumor types analyzed by whole genome, exome, or transcriptome sequencing, or acombination of these techniques,11-15 failed to identify any fusions involving these genes.As such, this fusion represents the first molecular feature unique to SFTs. These data alsosuggest that small molecule inhibitors of STAT6 may be efficacious in this tumor type.Further experiments investigating the functional behavior of this fusion protein are ongoing.Our estimates of the frequency of NAB2-STAT6 fusions should be considered a lower boundbecause whole exome sequencing would fail to identify intronic breaks, and because ourRT-PCR approach is limited to the regions covered.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe are grateful to the patients who contributed material for this study. We thank members of the GenomicsPlatform at the Broad Institute for their technical expertise, and Drs. Mohamed Abazeed and Austin Dulak forcritically reviewing the manuscript. This work was conducted as part of the Slim Initiative for Genomic Medicinein the Americas (SIGMA), a project funded by the Carlos Slim Health Institute in Mexico. This work wassupported by a SPORE grant in Soft Tissue Sarcoma (P50 CA140146) and a P01 grant (P01CA47179) to SamuelSinger (MSKCC). JC is supported by an American Cancer Society AstraZeneca Postdoctoral Fellowship; AMC issupported by a grant from the Kristen Ann Carr Foundation. MM is supported by generous donations from M.B.Zuckerman and Team Dragonfly of the Pan-Mass Challenge.References1. Mertens F, et al. Semin Oncol. 2009; 36:312–23. [PubMed: 19664492]2. Svaren J, et al. Mol Cell Biol. 1996; 16:3545–53. [PubMed: 8668170]3. Bhattacharyya S, et al. PLoS One. 2009; 4:e7620. [PubMed: 19888474]4. Shimoyamada H, et al. Am J Pathol. 2010; 177:70–83. [PubMed: 20489156]5. Abdulkadir SA, et al. Hum Pathol. 2001; 32:935–9. [PubMed: 11567222]6. Pal NR, Aguan K, Sharma A, Amari S. BMC Bioinformatics. 2007; 8:5. [PubMed: 17207284]7. Hebenstreit D, Wirnsberger G, Horejs-Hoeck J, Duschl A. Cytokine Growth Factor Rev. 2006;17:173–88. [PubMed: 16540365]8. Nelson EA, Sharma SV, Settleman J, Frank DA. Oncotarget. 2011; 2:518–24. [PubMed: 21680956]9. Merk BC, Owens JL, Lopes MB, Silva CM, Hussaini IM. BMC Cancer. 2011; 11:184. [PubMed:21595984]10. He HJ, et al. J Biol Chem. 2006; 281:31369–79. [PubMed: 16926159]11. Imielinski M, et al. Cell. 2012; 150:1107–20. [PubMed: 22980975]12. Hammerman PS, et al. Nature. 201213. Barbieri CE, et al. Nat Genet. 2012; 44:685–9. [PubMed: 22610119]14. Pugh TJ, et al. Nature. 2012; 488:106–10. [PubMed: 22820256]15. Banerji S, et al. Nature. 2012; 486:405–9. [PubMed: 22722202]Chmielecki et al. Page 3Nat Genet. Author manuscript; available in PMC 2014 April 11.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Structure of NAB2-STAT6(a) An inversion within chromosome 12 results in the juxtaposition of the NAB2 and STAT6genes. (b) Schematic diagram of five representative fusion variants showing a conservedbreakpoint within STAT6 and variable lengths of NAB2. White regions represent NAB2, redregions correspond to the 5′ untranslated region of STAT6, and blue regions show STAT6.Chmielecki et al. Page 4Nat Genet. Author manuscript; available in PMC 2014 April 11.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Whole exome sequencing identifies a recurrent NAB2-STAT6 fusion in solitary fibrous tumors

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Whole exome sequencing identifies a recurrent NAB2-STAT6 fusion in solitary fibrous tumors

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