Letters to the Editor 1173 Epigenetic disruption of two proapoptotic MAPK10/JNK3 and PTPN13/FAP-1 in multiple lymphomas and carcinomas through hypermethylation of a common bidirectional promoter

Leukemia (2006) 20, 1173–1175. doi:10.1038/sj.leu.2404193; node and peripheral blood mononuclear cell samples), both published online 30 March 2006 MAPK10 and PTPN13 were ubiquitously expressed, although at different levels (Figure 1c and d). Tumor suppressor genes can be inactivated through genetic or epigenetic mechanism, The Jun N-terminal (JNK) subgroup of the - or a combination of both mechanisms. As no homozygous activated protein kinase (MAPK) superfamily is implicated in deletion of both genes was found in all the cell lines examined, multiple signaling pathways of critical physiological processes, we speculated that their silencing might be mediated including , differentiation and proliferation.1 Stress- through epigenetic mechanism. We analyzed the methylation induced JNK activation leads to cell death through activation of status of their bidirectional promoter by methylation- the mitochondrial apoptotic pathway in many cell types and specific PCR (Table 1)8. Methylation was detected invirtually also to the interferon-alpha-induced apoptosis in B-cell lym- all the cell lines with reduced or silenced expression, and phoma.2 Some JNK member such as JNK1 can also promote the in some cell lines, methylation completely silenced the survival of BCR/ABL-transformed leukemia cells3 and is required expression of both genes (Figure 1c), which was further for the survival and proliferation of B-lymphoma cells.4 confirmed by high-resolution bisulfite genomic sequencing Frequent loss of heterozygosity (LOH) at specific chromo- analysis7 (Figure 1f). Treatment with the demethylating agent somal regions in human malignancies indicates the presence of 5-aza-20-deoxycitidine (Aza) induced robust expression of both candidate tumor suppressor genes (TSG) within the affected MAPK10 and PTPN13 in methylated cell lines (Figure 1g), regions. Loss of heterozygosity on the long arm of indicating that methylation directly mediates their silencing. 4 (4q) has been frequently reported in several tumors, including We also observed that in rare cell lines without MAPK10 hematopoietic malignancies.5,6 We have applied high-resolu- expression, no methylation was detected, indicating that other tion (1 Mb) array-comparative genomic hybridization (CGH)7 to mechanisms such as transcriptional repressors might also be detect genetic changes in 27 tumor cell lines (seven lymphoma infrequently involved. and 20 carcinoma cell lines), and identified a deletion at 4q21.3 We further evaluated the tumor suppressor function of in two esophageal carcinoma cell lines (Figure 1a). Two MAPK10 by transfecting MAPK10-expressing plasmid into proapoptotic proteins, MAPK10 (JNK3) and PTPN13 (PTPL1, silenced tumor cells.7 This ectopic expression of MAPK10 FAP-1), a protein tyrosine phosphatase, are located within the dramatically suppressed the colony formation of breast carci- deleted region, and share a 633-bp bidirectional promoter noma cells (Figure 1h), directly indicating that MAPK10 does which is a typical CpG island (Figure 1b). However, both genes function as a TSG. were not directly affected by the deletion. MAPK10 (JNK3) is a member of the JNK subgroup of MAP We further found that, in a total of 82 tumor cell lines, the kinase implicated in important physiological processes, includ- expression of these two genes was frequently downregulated ing apoptosis.9,10 Frequent loss of expression but no mutation of or silenced in non-Hodgkin’s lymphoma (94%, 15 of 16), MAPK10 in brain tumors has been reported, and MAPK10 Hodgkin’s lymphoma (50%, three of six), breast (30%, three of (JNK3) has been suggested as a class-II TSG being inactivated 10), gastric (60%, six of 10) and heptacellular (67%, eight of 12) epigenetically.11 In JNK3-deficient mice, the JNK3 signaling carcinoma cell lines (Figure 1c), whereas no downregulation pathway mediates cell apoptosis of the central nervous system.9 was detected in other carcinoma cell lines examined (Figure 1e). Disruption of JNK in murine fibroblasts resulted in the loss of In contrast, in all human normal tissues (including lymph tumor suppression in Ras-transformed cells, and the JNK

Table 1 PCR primer sequences and reaction conditions

PCR Primers Size (bp) TA (1C) PCR cycles

RT-PCR MAPK10 F: 50-cagctctctaaattgactcag-30 248 55 37 R: 50-ccaatgttggttcactgcag-30 PTPN13 F: 50-gcgctccagtagcaggac-30 191 55 37 R: 50-tcatctgtaaatgacacactac-30 GAPDH 33: 50-gatgaccttgcccacagcct-30 304 60 25 55: 50-atctctgcccctctgctga-30

Methylation-specific PCR (MSP) Methylated m3: 50-cgagtagttttagcggttac-30 160 58 40 m5: 50-aaaaccttctaacgcgaacga-30 Unmethylated u3: 50-tgtgagtagttttagtggttat-30 163 60 40 u5: 50-caaaaccttctaacacaaacaa-30

Bisulfite genomic sequencing (BGS) BGS3: 50-aggaggtggagttggatgtt-30 488 58 40 BGS2: 50-actaaaaaaactataaaaaaaaccta-30

TA, annealing temperature.

Leukemia Letters to the Editor 1174

Figure 1 (a) 1-Mb array-CGH identifies a 4q21.3 deletion in 2/27 tumor cell lines. (b) Structure of the bidirectional promoter of MAPK10 and PTPN13 with potential transcription factor (TF)-binding sites labeled. CpG sites are indicated by short vertical lines. (c) Representative analyses of the expression and methylation of MAPK10 and PTPN13 in multiple lymphoma and carcinoma cell lines. CGH, comparative genomic hybridization; M, methylated; U, unmethylated; BL, Burkitt’s lymphoma; DLBCL, diffuse large B-cell lymphoma; HL, Hodgkin’s lymphoma; HCC, hepatocellular carcinoma. (d) Expression of MAPK10 and PTPN13 in a panel of human adult normal tissues as detected by semi-quantitative reverse transcription-PCR, with GAPDH as a control. The tissues whose tumors showed frequent methylation were underlined. S. muscle, skeleton muscle; B.M., bone marrow; L.N., lymph node; PBMC, peripheral blood mononuclear cell. (e) No downregulation of MAPK10 and PTPN13 expression was found in nasopharyngeal carcinoma (NPC) and esophageal carcinoma (ESCC) cell lines . (f) Detailed mapping of CpG-methylation of the bidirectional promoter by bisulfite genomic sequencing (BGS). Each row of circles (CpG sites) represents an individual allele. BrCA, breast carcinoma; GsCA, gastric carcinoma. (g) Treatment with Aza activates MAPK10 and PTPN13 expression. (h) Ectopic expression of MAPK10 suppresses the colony formation of breast tumor cells. Monolayer culture using transient transfection was used.7 (i) Summary of the frequencies of promoter methylation in various tumor cell lines. Ca, carcinoma.

pathway contributes to the elimination of transformed cells sitol3-kinase/Akt pathway,13 and its mutations have been through apoptosis in vivo.12 PTPN13 also induces Fas-independent detected in tumors.14 Thus, our results together with previous apoptosis in breast cancer cells, through the phosphatidylino- studies suggest that both MAPK10 (JNK3), different from other

Leukemia Letter to the editor 1175 JNK member (JNK1, JNK2),4 and PTPN13 have proapoptotic 5 Viguie F, Aboura A, Bouscary D, Ramond S, Delmer A, Tachdjian G functions and can function as TSGs to suppress tumorigenesis. et al. Common 4q24 deletion in four cases of hematopoietic However, their tumor suppressor functions were frequently malignancy: early stem cell involvement? Leukemia 2005; 19: disrupted epigenetically in multiple lymphomas and carcinomas 1411–1415. 6 Garcia JL, Hernandez JM, Gutierrez NC, Flores T, Gonzalez D, owing to the methylation of their shared bidirectional promoter Calasanz MJ et al. Abnormalities on 1q and 7q are associated with (Figure 1i). poor outcome in sporadic Burkitt’s lymphoma. A cytogenetic and comparative genomic hybridization study. Leukemia 2003; 17: 2016–2024. Acknowledgements 7 Ying J, Li H, Seng TJ, Langford C, Srivastava G, Tsao SW et al. Functional epigenetics identifies a protocadherin PCDH10 as a We thank Dr Tzer Jing Seng for helping the array-CGH, Dr candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene Riccardo Dalla-Favera for DLBCL cell lines and Dr Roger J Davis 2006; 25: 1070–1080. for the full-length MAPK10 cDNA clone. This study was supported 8 Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. by a Michael Kadoorie Cancer Genetics Research Fund grant. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: J Ying1,HLi1, Y Cui1, AHY Wong1, C Langford2 and Q Tao1 9821–9826. 1Department of Clinical Oncology, Cancer Center, 9 Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ Chinese University of Hong Kong, Hong Kong and et al. Absence of excitotoxicity-induced apoptosis in the 2Wellcome Trust Sanger Institute, Cambridge, UK hippocampus of mice lacking the Jnk3 . Nature 1997; 389: E-mail: [email protected] 865–870. 10 Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000; 288: References 870–874. 11 Yoshida S, Fukino K, Harada H, Nagai H, Imoto I, Inazawa J et al. 1 Davis RJ. Signal transduction by the JNK group of MAP . Cell The c-Jun NH2-terminal kinase3 (JNK3) gene: genomic structure, 2000; 103: 239–252. chromosomal assignment, and loss of expression in brain tumors. 2 Yanase N, Hata K, Shimo K, Hayashida M, Evers BM, Mizuguchi J. J Hum Genet 2001; 46: 182–187. Requirement of c-Jun NH2-terminal kinase activation in interferon- 12 Kennedy NJ, Sluss HK, Jones SN, Bar-Sagi D, Flavell RA, Davis RJ. alpha-induced apoptosis through upregulation of tumor necrosis Suppression of Ras-stimulated transformation by the JNK signal factor-related apoptosis-inducing ligand (TRAIL) in Daudi B transduction pathway. Genes Dev 2003; 17: 629–637. lymphoma cells. Exp Cell Res 2005; 310: 10–21. 13 Bompard G, Puech C, Prebois C, Vignon F, Freiss G. 3 Hess P, Pihan G, Sawyers CL, Flavell RA, Davis RJ. Survival Protein-tyrosine phosphatase PTPL1/FAP-1 triggers apoptosis signaling mediated by c-Jun NH(2)-terminal kinase in transformed B in human breast cancer cells. J Biol Chem 2002; 277: lymphoblasts. Nat Genet 2002; 32: 201–205. 47861–47869. 4 Gururajan M, Chui R, Karuppannan AK, Ke J, Jennings CD, Bondada 14 Wang Z, Shen D, Parsons DW, Bardelli A, Sager J, Szabo S et al. S. c-Jun N-terminal kinase (JNK) is required for survival and Mutational analysis of the tyrosine phosphatome in colorectal proliferation of B-lymphoma cells. Blood 2005; 106: 1382–1391. cancers. Science 2004; 304: 1164–1166.

t(5;12)(q23–31;p13) with ETV6-ACSL6 gene fusion in polycythemia vera

Leukemia (2006) 20, 1175–1178. doi:10.1038/sj.leu.2404194; Patient C1, a 29-year-old man, presented in October 2002 with published online 30 March 2006 midly enlarged liver and spleen, effort dyspnea, and polycythe- mia with 59.7% hematocrit level. The bone marrow (BM) was Myeloproliferative disorders (MPD) are chronic clonal prolifera- hypercellular, with rare dystrophic megakaryocytes and 6% tions of hematopoietic progenitors. Typical MPDs include chronic eosinophils, without basophilia. Isotopic exploration showed an myeloid leukemia (CML), polycythemia vera (PV), essential increased total red blood cell mass. Cytogenetic analysis with thrombocythemia (ET) and idiopathic myelofibrosis (IF). Onco- R-banding technique on BM cells detected a t(5;12)(q23–31;p13) genic alterations identified so far in MPDs target tyrosine kinases, translocation in three metaphases among 24 analyzed (13%) result from chromosomal translocations or gene mutations and (Table 1). The patient was treated by phlebotomy but dyspnea lead to constitutive activation of survival and proliferation persisted, and prurit, paresthesia and headaches appeared. pathways. Reciprocal translocations lead to gene fusion and Three months later, a treatment by hydroxyurea was started. production of chimeric proteins such as BCR-ABL, ETV6-PDGFRB Bone marrow showed dystrophic megakaryocytes of the PV or PCM1-JAK2.1,2 Point mutations of the JAK2 kinase occur in type. In May 2005, BM analysis showed increased eosinophilia almost all PV, and in around half of ET and IF.3 JAK2 functions (16%) and basophilia (10%), and on karyotype, four metaphases downstream of membrane receptors, including cytokine receptors among 20 (20%) showed the t(5;12) translocation. In January such as interleukin-3 (IL3) receptors. Overproduction of IL3 has 2006, 3.5 years after diagnosis, the patient is alive under been reported in atypical CML following rearrangements of the hydroxyurea treatment, the white blood cell count is 9.6 Â 109/l IL3 gene upstream region in cells from patients with t(5;12)(q23– with 15% eosinophils and 15% basophils, hemoglobin level 31;p13) translocation and ETV6-ACSL6 fusion.4–7 16.5 and platelet count 484 Â 109/l. The indication of allogenic We report here two cases of t(5;12)(q23–31;p13) translocation transplantation is under discussion. with ETV6-ACSL6 rearrangement in PV patients. The main Patient C2, a 31-year-old woman, presented in 1979 with PV. characteristics of the two patients (designated C1, C2) are She was treated by phlebotomy and splenic irradiation. In 1998, reported in Table 1, along with the cases reported so far.4–7 because of an important splenomegaly, a second splenic

Leukemia