Leukemia (2009) 23, 2153–2186 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu LETTERS TO THE EDITOR

Biallelic inactivation of TRAF3 in a subset of B-cell lymphomas with interstitial del(14)(q24.1q32.33)

Leukemia (2009) 23, 2153–2155; doi:10.1038/leu.2009.149; hypothesis is that the deletion might inactivate a tumor published online 20 August 2009 suppressor . Among many other , the TNF receptor-associated factor The interstitial deletion del(14)(q24.1q32.33) is a recurrent 3(TRAF3)-gene in 14q32.32 is located within the deleted region. chromosomal change in B-cell chronic lymphocytic leukemia The TRAF3 negatively regulates the NF-kB pathway and (CLL) and other B-cell lymphomas.1,2 The break points of this has been shown to be recurrently affected by biallelic TRAF3 deletion are located in the immunoglobulin heavy chain (IGH)- deletion and inactivating mutation in multiple myeloma and locus in 14q32.33 and in a region in 14q24.1 containing the Waldenstro¨m’s macroglobulinemia.3–5 Furthermore, B-cell- ZFP36L1 gene (1and our own unpublished observations). specific TRAF3À/À mice show remarkably prolonged B-cell Activation of a candidate oncogene centromeric of the deletion survival and highly expanded B-cell compartments in secondary break point in 14q24.1 through juxtaposition to the IGH locus, lymphoid organs.6 These facts qualify TRAF3 as a candidate yet could not be unambiguously shown.1 An alternative tumor suppressor gene involved in lymphomagenesis.

+2 +1 0 -1 -2

101.1Mb 101.5Mb 101.9Mb 102.3Mb 102.7Mb 103.2Mb 103.6Mb 14

+2 +1 0 -1 -2

q12 q13.2q21.1 q21.3 q22.2 q23.1 q23.3 q24.2 q31.1 q31.3 q32.2 q32.12 q32.32

Exons 3 4 5 6 789 10 11 12 0.0008

2.5

0.0 1aa RING finger Zincfingers Coiled-Coil MATH / TRAF 568 aa Case 7 -2.5

-5.0 Case 8 fold change

Case 8 -7.5

Case 9 -10.0 14q- CLL CLL

Figure 1 FISH, array CGH, expression and mutation analyses of the TRAF3 gene in cases with deletion del(14)(q24.1q32.33). (a) Interphase FISH on case 1 using the TRAF3 FISH assay. The green-labeled BAC clone CTD-3235K2 (green) hybridizes to the TRAF3 locus, the red-labeled BAC clone RP11-977G1 (red) serves as internal control and hybridizes outside the heterozygously deleted region on 14 (Supplementary Table 1). The upper nucleus harbors a homozygous deletion of CTD-3235K2, the left nucleus indicates a normal hybridization pattern and the right nucleus shows a heterozygous deletion of the TRAF3 locus. (b) Array CGH of case 2 performed on the CGH microarray 244A platform (Agilent) showing the heterozygously and the homozygously deleted region around TRAF3 in the long arm of . A value of zero indicates a balanced chromosome status. (c) Schematic illustration of the genomic organization of the coding exons of TRAF3 (exons 3–12) and the encoded protein domains. The non-coding exons 1 and 2 have not been analyzed. Beneath, a graphic display of the four mutated predicted in three cases is shown. : missense mutation and stop codon; : putative splice site mutation; : point mutation; aa: amino acids. (d) Scatter plot comparing the relative TRAF3–RNA expression of CLLs with (left) and without (right) (case 9) deletion del(14)(q24.1q32.33) as determined by quantitative RT-PCR. (P-value ¼ 0.0008, Mann–Whitney test). Letters to the Editor 2154 Here, we studied the TRAF3 gene in 30 chronic lymphocytic leukemias (CLLs) and 11 B-cell lymphomas of different subtypes

including marginal zone lymphoma and B-cell lymphoma not 15 otherwise specified. All cases carried a 14q-deletion with the typical break points in the ZFP36L1 region in 14q24.1 and the IGH locus in 14q32.33 as shown by fluorescence in situ

hybridization (FISH). By FISH we screened the 41 cases for +putative splice site homozygous deletions in the TRAF3 region using a dual-color FISH assay (BAC clone CTD-3235K2 for the TRAF3 locus and BAC clone RP11-977G1 as internal control, Supplementary

Information). We identified two cases (case 1 and 2) harboring p.Cys53Asp p.Lys99fs c.288_297+6del homozygous deletions of the TRAF3 locus in 62 and 76% of the - A] -

cells, respectively. In addition, case 1 showed a signal pattern 4 indicating a heterozygous deletion of the TRAF3 region in 14% therwise specified; SMZL, splenic marginal

of the nuclei suggesting that the homozygous deletion was putative splice site mutation (Exon 4/Intron) (DNA change and predicted protein change) p.Ser223fs - a secondary event (Figure 1a). High resolution array CGH b (3/7 clones) - (Agilent, 244 K and NimbleGen, 385 K) confirmed the homo- C; 820-1G zygous deletions and sized them to approximately 690 kb 4 (chr14:101 988 kb–102 675 kb) and 670 kb (chr14:102 000 kb– Aberration c.669delA mutation (Intron/Exon 10) c.[157T c.296delA 102 669 kb) (Figure 1b) (Supplementary Information). Remark- (2/7 clones) ably, the centromeric and the telomeric break points of both homozygous deletions cluster within 6 and 12 kb in the regions of the genes KIAA0329 and TNFAIP2, respectively. Furthermore, FISH identified four cases (cases 3–6) with homozygous deletions of TRAF3 in subclones with 7–14% of the nuclei (cutoff for homozygous loss: o1%). 1) Subclonal homozygous deletion (FISH, array CGH) 1) Subclonal homozygous deletion (FISH) 1) Subclonal homozygous deletion (FISH) 1) Subclonal homozygous deletion (FISH) 1) Subclonal homozygous deletion (FISH)   To investigate whether the remaining TRAF3 allele in the   cases with heterozygous TRAF3 deletion could be inactivated by  mutations, the 10 coding exons of TRAF3 (exon 3–12; 0); 14 ( 0) Homozygous deletion (FISH, array CGH) 0); 68 ( 0); 76 ( 1) 1) 1) 0); 82 ( NM_145725) were screened by denaturing high performance 0); 37 (         liquid chromatography. PCR products with aberrant denaturing  Percentage of aberrant nucleiFISH by (number of TRAF3 copies) 62 ( 64 ( 93 ( 89 ( high performance liquid chromatography chromatogram were 80 ( subjected to sequence analysis (Supplementary Information, Supplementary Table 2). From 31 del(14)(q24.1q32.33)-positive cases with available DNA and a tumor cell content of X35%, three cases showed a total of five mutations presumably disturbing the TRAF3 function (Supplementary Figure 1). These del(14)(q22) comprise two frameshift mutations leading to truncated proteins, isoform 1) was used.

two splice site mutations and one missense mutation affecting [19]/46,XY[1] 11 ( [14]. 7 ( the consensus sequence of the ring finger motif (Figure 1c).7 [23]/46,XY[7] 14 ( TRAF3 [5]/46,XY[15] 7 (

There is no evidence for polymorphisms at these DNA sequence del(14)(q22q32) positions in the Human Genome Browser database (http:// genome.ucsc.edu/) and denaturing high performance liquid ,-20,der(?)t(?;2)(?;p13), chromatography or sequencing analyses failed to detect these del(14)(q22) del(14)(q22-q23) del(14)(q22)

five alterations in at least 50 DNA samples derived from a del(14)(q21q32.33) del(14)(q11?q24?) peripheral blood of healthy individuals. The cases with biallelic del(14)(q22q32)

TRAF3 aberrations are summarized in Table 1. del(14)(q22) TRAF3 mRNA expression determined by quantitative reverse Karyotype -14, der(?)t(?;8)(?;q21.1) PCR in nine del(14)(q24.1q32.33)-positive cases without homozygous TRAF3 deletion was compared with that in 11 B-CLLs without TRAF3-aberration by FISH (Supplementary

Information). All cases had a tumor cell content of more than (years) 40%. Cases with TRAF3 deletion showed significantly lower diagnosis expression of TRAF3 mRNA compared with B-CLLs without TRAF3 deletion (P ¼ 0.0008)(Figure 1d). To investigate whether low expression of TRAF3 in cases with heterozygous 14q-deletion might be because of aberrant promoter methylation, we performed methylation-specific PCR in 32 cases with del(14)(q24.1q32.33) (Supplementary Informa- aberrations in lymphomas with deletion del(14)(q24.1q32.33) tion). Though no CpG-island has been assigned to TRAF3,it

contains a highly conserved, CpG-rich region within the TRAF3 promoter region (http://genome.ucsc.edu/cgi-bin/hgGateway). We established two methylation-specific PCR assays, one targets

a region located 58 bp upstream of the transcription start site in 14q-alterations are indicated in bold letters. Case Diagnose Sex Age at Table 1 a,b 1 CLL M 49 45,XY,add(2)(p13),del(7)(q11),add(12)(q24), 2 SMZL M 82 46,XY,t(5;7)(p15;p11), 3 MCL M 52 46,XY, 4 lymphoma NOS F 85 46,XX, 5 MZL M 74 46,XY, 6 CLL M 70 47,XY,+12, 7 Low-grade NHL M 55 47,XY,+der(12)t(12;12)(p11;p13), 8 CLL M 61 47,XY,+12, zone lymphoma. For description of genomic variants, the reference sequence NM_145725 ( Abbreviations: CLL, chronic lymphocytic leukemia; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; NHL, non-Hodgkin’s lymphoma; NOS, not o the first non-coding exon of TRAF3, whereas the other one 9 CLL F 54 47,XX,+12,

Leukemia Letters to the Editor 2155 targets a conserved E2F transcription factor-binding site 546 bp JA Martinez-Climent4, C Bastard5, M Salido6, E. Schroers7, 1 1 1 8 upstream of the transcription start site (Supplementary Table 3). JI Martin-Subero , S Gesk , L Harder , A Majid , MJS Dyer8 and R Siebert1 All 32 cases analyzed showed the pattern for unmethylated 1 DNA like the controls (data not shown). Institute of Human Genetics, Christian-Albrechts University Kiel and University Hospital Schleswig-Holstein, Kiel, In this study, we identified biallelic inactivation of TRAF3 Germany; (9/41, 22%) and lower expression of TRAF3 mRNA in B-cell 2Laboratoire d’Hematologie et de cytoge´ne´tique, Centre neoplasias with deletion del(14)(q24.1q32.33) due to breaks in Hospitalier Lyon Sud, Lyon, France; IGH and ZFP36L1 regions. Abnormalities that inactivate the 3Department of Genetics, University of Navarra, TRAF3 protein lead to constitutive NF-kB activation,4,6 which is Pamplona, Spain; a known mechanism in various cancer types to block 4Division of Oncology, Center for Applied Medical Research, apoptosis.8 The ring finger domain and the TRAF3 domain are University of Navarra, Pamplona, Spain; 5 the critical components of TRAF3 for the suppression of NIK Groupe d’Etude des Prolife´rations Lymphoides (GPL), protein levels and the subsequent inhibition of NF-kB2.7 At least European Institute for Peptides Research (IFRMP23), Centre Henri Becquerel, Rouen, France; one of the two domains is affected in each of the three TRAF3- 6Servei de Patologia, Laboratori de Citogene`tica Molecular, mutated alleles, suggesting TRAF3 inactivation in these cases. Hospital del Mar, GRETNHE, IMIM, Barcelona, Spain; The fact that we observed cases with subclonal homozygous 7MVZ Dr Eberhard and Partner, Dortmund, Germany; deletion of the second TRAF3 allele indicates that this loss 8Medical Research Council (MRC) Toxicology Unit, University occurred secondary during tumor progression. It is still possible of Leicester, Leicester, UK that oncogene activation through juxtaposition of IGH in E-mail: [email protected] 14q32.33 to the chromosomal region in 14q24.1 is the major mechanism in lymphomagenesis conferred by the deletion del(14)(q24.1q32.33). Complete loss of functional TRAF3 by inactivation of the remaining allele may induce a further References selective advantage of the tumor cells. B-cell-specific TRAF3À/À mice show an increased production of immunoglobulins.7 1 Pospisilova H, Baens M, Michaux L, Stul M, Van Hummelen P, Van Therefore, it is intriguing to speculate that loss of functional Loo P et al. Interstitial del(14)(q) involving IGH: a novel recurrent TRAF3 in cases with deletion del(14)(q24.1q32.33) leads to aberration in B-NHL. Leukemia 2007; 21: 2079–2083. 2 Tilly H, Bastard C, Halkin E, Lenormand B, Bizet M, Dauce JP et al. stronger activation of the immunoglobulin enhancer and, Del(14)(q22) in diffuse B-cell lymphocytic lymphoma. Am J Clin subsequently, a stronger activation of a putative oncogene in Pathol 1988; 89: 109–113. 14q24 that is juxtaposed to the IGH locus. 3 Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, Zhan F et al. Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple Conflict of interest myeloma. Cancer Cell 2007; 12: 115–130. 4 Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng WJ et al. Promiscuous mutations activate the noncanonical NF-kappaB The authors declare no conflict of interest. pathway in multiple myeloma. Cancer Cell 2007; 12: 131–144. 5 Braggio E, Keats JJ, Leleu X, Van Wier S, Jimenez-Zepeda VH, Valdez R et al. Identification of copy number abnormalities and Acknowledgements inactivating mutations in two negative regulators of nuclear factor- kappaB signaling pathways in Waldenstrom0s macroglobulinemia. We gratefully thank Kerstin Runde, Reina Zu¨hlke-Jenisch, Magret Cancer Res 2009; 69: 3579–3588. Ratjen, Dorit Schuster, Claudia Becher and Ursula Schnaidt for 6 Xie P, Stunz LL, Larison KD, Yang B, Bishop GA. Tumor necrosis their technical assistance. Supported by the Network Project of the factor receptor-associated factor 3 is a critical regulator of B cell Deutsche Krebshilfe ‘Molecular Mechanisms in Malignant homeostasis in secondary lymphoid organs. Immunity 2007; 27: 253–267. Lymphomas’ 70-3173-TR3, the Wilhelm Sander-Stiftung 2005.168.2 7 He JQ, Saha SK, Kang JR, Zarnegar B, Cheng G. Specificity of TRAF3 and RETICS. Instituto de Salud Carlos III, RETICS. Convocatoria in its negative regulation of the noncanonical NF-kappa B pathway. 2008. RD07/0020/2004. J Biol Chem 2007; 282: 3688–3694. 8 Basseres DS, Baldwin AS. Nuclear factor-kappaB and inhibitor of 1 1 1 1 1 I Nagel , S Bug ,HTo¨nnies , O Ammerpohl , J Richter , kappaB kinase pathways in oncogenic initiation and progression. I Vater1, E Callet-Bauchu2, MJ Calasanz3, Oncogene 2006; 25: 6817–6830.

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

Imatinib mesylate causes growth plate closure in vivo

Leukemia (2009) 23, 2155–2159; doi:10.1038/leu.2009.150; BCR–Abl,1 stem cell factor receptor (c-Kit),2 platelet-derived published online 23 July 2009 receptor (PDGFR)-a and PDGFR-b1 and the macrophage colony-stimulating factor receptor, c-fms (CSF-1R).3 Imatinib mesylate (Glivec/Gleevec, STI571; Novartis, Basel, Imatinib is a highly effective treatment for Philadelphia chromo- Switzerland) is a 2-phenylaminopyrimidine-based ATP-compe- some-positive chronic myeloid leukaemia (CML) and gastro- titive protein tyrosine kinase inhibitor, which at pharmaco- intestinal stromal tumours, based on the inhibition of BCR–Abl logically-achievable concentrations inhibits the activity of and c-Kit, respectively. Although imatinib is the gold standard

Leukemia