Inhibition of Wnt16 in human acute lymphoblastoid leukemia cells containing the t(1;19) translocation induces apoptosis Julien Mazieres 1,2 , Liang You 1 , Biao He 1 , Zhidong Xu 1 , Amie Y Lee 1 , Iwao Mikami 1 , Frank McCormick 1 and David M Jablons* ,1 1 UCSF Comprehensive Cancer Center, San Francisco, CA 94115, USA; 2 Department Innovation The´rapeutique et Oncologie Mole´culaire, INSERM U563, Institut Claudius Regaud, 31052 Toulouse Cedex, France The Wnt family of secreted glycoproteins is widely involved in cell proliferation, differentiation and oncogen- esis. Many Wnt signaling genes are upregulated and activated in chronic lymphocytic leukemia. Less is known concerning acute leukemia. One subtype of acute lymphoblastoid leukemia (ALL) is characterized by a t(1;19) chromosomal translocation resulting in a fusion protein E2A–Pbx1 that promotes transformation and leukemogenesis. Wnt16 has been shown to be targeted by E2A–Pbx1. We performed a differential gene expression array in acute leukemia cell lines displaying or not displaying the t(1;19) translocation. We found that Wnt16 and many Wnt signaling-related genes were upregulated in the translocation-containing cells. As two isoforms of Wnt16, Wnt16a and Wnt16b, have been recently identi- fied, we demonstrated by using RT–PCR and Western blot that Wnt16b (and not Wnt16a) is overexpressed in t(1;19)-containing cell lines. We then directly addressed the role played by both isoforms in this type of leukemia. Using specific short interfering RNA (siRNA) and an anti- Wnt16 antibody, we showed that targeted-Wnt16b inhibi- tion leads to apoptotic cell death. We also demonstrated that Wnt16b mediates its effect through the canonical Wnt pathway involving dishevelled-2, b-catenin and survivin. We thus propose that Wnt16 plays an important role in leukemogenesis, raising its therapeutic interest. Oncogene (2005) 24, 5396–5400.doi:10.1038/sj.onc.1208568; published online 20 June 2005 Keywords: Wnt; Wnt16; leukemia; apoptosis; human antibody; RNA interference The Wnt family of secreted glycoproteins is a group of signaling molecules that is widely involved in develop- mental processes and oncogenesis. Currently, 19 human Wnt proteins have been identified. The proto-oncogenic effects of Wnt were discovered more than 20 years ago (Nusse and Varmus, 1982) and since then numerous reports have demonstrated aberrant activation of the Wnt signaling pathway in disparate human cancers (Morin et al., 1997; Rhee et al., 2002; Weeraratna et al., 2002). We reported the overexpression of dishevelled (Dvl) proteins in thoracic malignancies (Uematsu et al., 2003a, b). It is also known that Wnt proteins regulate early B-cell growth and survival (Reya et al., 2003) and the activation of the Wnt signaling pathway has been demonstrated in chronic lymphocytic leukemia (Lu et al., 2004). AnotherareaofinterestregardingtheWntproteinsis the acute leukemias that are often characterized by translocations that can determine prognosis and treat- ment options. Muller-Tidow et al. (2004) reported that translocations in acute myeloid leukemia could activate the Wnt signaling pathway. Among these chromosomal abnormalities, the t(1;19) translocation results in the production of chimeric E2A–Pbx1 proteins that display oncogenic properties (Kamps et al., 1991). The mole- cular properties of E2A–Pbx1 have been extensively characterized and several candidate target genes have been identified. Light has been shed on Wnt16 in recent studies.Representationaldifferentialexpressionanalysis performed in leukemia cell lines containing the fusion protein vs others lacking E2A–Pbx1 identified Wnt16 as a putative target gene (McWhirter et al., 1999). More- over, gene expression profiling of leukemic blasts showed a marked overexpression of Wnt16 in E2A– Pbx1-expressing leukemias (Ross et al., 2003). Nevertheless, the specific role played by Wnt16 and the Wnt-related proteins in acute leukemia has never been addressed so far. Moreover, Fear et al. identified two isoforms of Wnt16, Wnt16a and Wnt16b, and the respective role of each isoform in oncogenesis has also yet to be elucidated. In order to analyse the Wnt signaling pathway activation, we first performed gene expression profiling using a custom array designed to profile the expression oftheWntsignalingpathway-relatedgenes(GEArrayQ Human Wnt Signaling Pathway Gene Array, Super- Array). Four acute lymphoblastic leukemia cell lines were analysed, two containing the t(1;19) translocation (697andRCH-ACV)andtwowithoutthetranslocation (CCL-119andNALM-6).WefirstconfirmedtheWnt16 overexpression in both 697 and RCH-ACV cell lines, whereas no signal was seen in the control cell lines. Received 28 July 2004; revised 17 January 2005; accepted 24 January 2005; published online 20 June 2005 *Correspondence: DM Jablons, Department of Surgery, Cancer Center, 1600 Divisadero St, C322C, Box 1674, San Francisco, CA 94115, USA; E-mail: jablonsd@surgery.ucsf.edu Oncogene (2005) 24, 5396–5400 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc