Characterization of the chronic myelomonocytic leukemia associated TEL-PDGFbR fusion protein Tobias SjoÈblom 1,3 , Anthony Boureux 2,3 , Lars RoÈnnstrand 1 , Carl-Henrik Heldin 1 , Jacques Ghysdael 2 and Arne O È stman* ,1 1 Ludwig Institute for Cancer Research, Box 595, S-751 24, Uppsala, Sweden; 2 CNRS UMR 146, Institut Curie-Section de RecheÁrche, Centre Universitaire, 91405 Orsay, France The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGFbR, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF b-receptor (PDGFbR). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGFbR and also indicated that the TEL moiety activates the tyrosine kinase of the PDGFbR through the formation of TEL-PDGFbR oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGFbR, suggesting a strategy for antagonizing the signaling of TEL-PDGFbR, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGFbR and ligand-stimulated PDGFbR revealed that only TEL- PDGFbR expression conferred IL-3-independent growth, suggesting dierences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL- PDGFbR was identi®ed as autophosphorylation sites in TEL-PDGFbR. Keywords: chronic myelomonocytic leukemia; chromo- somal translocation; platelet-derived growth factor; TEL Introduction Chromosomal rearrangements leading to fusion of protein oligomerization domains to tyrosine kinase domains occur in a number of malignancies (Rabbitts, 1994; Sawyers and Denny, 1994). The t(5;12) translocation, associated with chronic myelomonocytic leukemia (CMML), generates a fusion gene encoding a protein composed of the 154 amino-terminal amino acids of TEL and the transmembrane and intracellular tyrosine kinase domain of the PDGF b-receptor (PDGFbR) (Golub et al., 1994). The PDGFbR is composed of an extracellular part, consisting of ®ve Ig-like domains, a transmembrane segment and an intracellular part containing a split tyrosine kinase domain. PDGF-BB, the ligand of the PDGFbR, induces activation of PDGFbR through dimerization of receptors. Dimerization of receptors leads to activation of the intrinsic kinase activity and subsequent autophosphorylation of tyrosine residues in the intracellular part, which serve as docking sites for SH-2-domain-containing signaling proteins (Heldin et al., 1998). Dysregulated activation of the PDGFbR, through ectopic ligand production, has been suggested to contribute to malignant cell growth in a number of dierent tumor types, including glioblastomas, me- sotheliomas and sarcomas (reviewed in Heldin and Westermark, 1996). Also, in addition to the TEL- PDGFbR fusion protein, two other genetic rearrange- ments giving rise to fusion proteins containing the kinase domain of the PDGFbR has recently been identi®ed; the CEV14-PDGFbR and HIP-PDGFbR genes isolated from an AML and a CMML patient, respectively (Abe et al., 1997; Ross et al., 1998). TEL, also known as ETV6, is a 452 amino acid residue protein of the ETS family of sequence speci®c transcription factors (Ghysdael and Boureux, 1997). The ETS family members contain a conserved DNA binding domain, the ETS domain. A subset of ETS proteins, including TEL, also contains a conserved amino-terminal domain termed the pointed domain/B domain. This domain has been shown to be necessary and sucient for homophilic self-association of TEL- PDGFbR (Carroll et al., 1996; Jousset et al., 1997). Malignancy-associated chromosomal rearrangements leading to fusion of the pointed/B domain of TEL to a number of other tyrosine kinases, including c-ABL, JAK2 and TRK-C, have recently been characterized (Golub et al., 1995; Knezevich et al., 1998; Lacronique et al., 1997; Papadopoulos et al., 1995). In vitro and in vivo transforming ability of TEL-PDGFbR and TEL- JAK2 has been demonstrated (Carroll et al., 1996; Jousset et al., 1997; Schwaller et al., 1998; Thomasson et al., 1999). TEL has also been described as a fusion partner in human leukemias for other transcriptional regulators like AML1 and CDX2 (Chase et al., 1999; Golub et al., 1995; Romana et al., 1995). The aim of the present study was to further investigate the importance of TEL-mediated oligomer- ization for the activation of TEL-PDGFbR and to compare the signaling properties of TEL-PDGFbR and ligand-activated PDGFbR. We show that over- expression of the TEL moiety leads to reduction of tyrosine phosphorylation of the fusion protein. We also demonstrate dierences in the signaling capacity between ligand-stimulated PDGFbR and TEL- PDGFbR and provide evidence for the presence of unique autophosphorylation sites in TEL-PDGFbR. *Correspondence: A O È stman 3 The ®rst two authors contributed equally to this paper Received 6 May 1999; revised 26 August 1999; accepted 1 September 1999 Oncogene (1999) 18, 7055 ± 7062 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc