Oncogene (2004) 23, 2287–2297 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc ORIGINAL PAPERS Frameshift mutation in the Dok1 gene in chronic lymphocytic leukemia Sanghoon Lee1, Franc¸ois Roy1, Carlos M Galmarini2, Rosita Accardi1, Jocelyne Michelon1, Alexandra Viller1, Emeline Cros2, Charles Dumontet2 and Bakary S Sylla*,1 1International Agency for Research on Cancer, 150 Cours Albert-Thomas, Lyon 69008, France; and 2INSERM U 590 and Hospices Civils de Lyon, Lyon 69008, France B-cell chronic lymphocytic leukemia (B-CLL) is a mulation of monoclonal CD5 þ , CD23 þ mature B malignant disease characterized by an accumulation of cells, with a high percentage of cells arrested in the G0/ monoclonal CD5 þ mature Bcells, with a high percentage G1 phase of the cell cycle. These cells have a severe of cells arrested in the G0/G1 phase of the cell cycle, and a defect in programed cell death due to high expression of particular resistance toward apoptosis-inducing agents. antiapoptotic proteins, like bcl-2 family proteins bcl-2, Dok1 (downstream of tyrosine kinases) is an abundant bcl-xL, bax and mcl-1 or survival factors such as Ras-GTPase-activating protein (Ras-GAP)-associated TRAF1, and TRAF2 (Schroeder and Dighiero, 1994; tyrosine kinase substrate, which negatively regulates cell Kitada et al., 1998; Caligaris-Cappio and Hamblin, proliferation, downregulates MAP kinase activation and 1999; Munzert et al., 2002). Signaling pathways such as promotes cell migration. The gene encoding Dok1 maps to signal transducer and activator of transcription 1 (STAT human chromosome 2p13, a region previously found to be 1) and (STAT 3), phosphatidylinositol-3 kinase (PI-3K) rearranged in B-CLL. We have screened the Dok1 gene or NF-kB have been found constitutively activated in B- for mutations from 46 individuals with B-CLL using CLL cells (Frank et al., 1997; Furman et al., 2000; heteroduplex analysis. A four-nucleotide GGCC deletion Munzert et al., 2002; Ringshausen et al., 2002). in the coding region was found in the leukemia cells from Activation of such pathways may contribute to the one patient. This mutation causes a frameshift leading to disease. protein truncation at the carboxyl-terminus, with the So far, no recurrent gene mutation has been found acquisition of a novel amino-acid sequence. In contrast to in B-CLL. Instead, numerous chromosomal abnor- the wild-type Dok1 protein, which has cytoplasmic/ malities involving chromosomes 6q21–q23, 11q22.3– membrane localization, the mutant Dok1 is a nuclear q23.1, 13q14, 17p13 or 2p13 have been identified, protein containing a functional bipartite nuclear localiza- some of which have clinical significance (Nelms et al., tion signal. Whereas overexpression of wild-type Dok1 1998; Wyatt et al., 2000). The p53 tumor-suppressor inhibited PDGF-induced MAP kinase activation, this gene and the ATM gene have been found mutated in inhibition was not observed with the mutant Dok1. B-CLL and p53 mutations are associated with Furthermore the mutant Dok1 forms heterodimers with resistance to chemotherapy (El Rouby et al., 1993; Dok1 wild type and the association can be enhanced by Wattel et al., 1994; Schaffner et al., 1999; Stankovic Lck-mediated tyrosine-phosphorylation. This is the first et al., 1999). example of a Dok1 mutation in B-CLL and the data Dok1 (downstream of tyrosine kinases), or p62Dok1, suggest that Dok1 might play a role in leukemogenesis. is a phosphorylated protein downstream of a wide range Oncogene (2004) 23, 2287–2297. doi:10.1038/sj.onc.1207385 of protein-tyrosine kinases (PTKs) (Carpino et al., 1997; Published online 19 January 2004 Yamanashi and Baltimore, 1997; Bhat et al., 1998). Dok1 and the related proteins Dok2, Dok3, Dok4, Keywords: Dok1; chronic lymphocytic leukemia; frame- Dok5 and the insulin receptor substrate (IRS) (Nelms shift mutation; NLS; Lck; tyrosine phosphorylation et al., 1998; White, 1998; Cong et al., 1999; Lemay et al., 2000; Di Cristofano et al., 2001; Grimm et al., 2001; Cai et al., 2003; Favre et al., 2003) contain a pleckstrin homology (PH) domain at the N-terminus, a central phosphotyrosine-binding (PTB) domain and a C-term- Introduction inal domain rich in proline and several tyrosine- phosphorylation sites (Carpino et al., 1997; Yamanashi B-cell chronic lymphocytic leukemia (B-CLL) is the and Baltimore, 1997). After tyrosine-phosphorylation, most common type of adult leukemia found in Western Dok1 can interact with other signaling molecules populations. The disease is characterized by an accu- containing SH2 domains, such as Ras-GTPase-activat- ing protein (Ras-GAP), SHIP1, Nck, Csk and the X-linked lymphoproliferative syndrome (XLP) gene *Correspondence: BS Sylla; E-mail: [email protected] Received 23 May 2003; revised 19 November 2003; accepted 20 product SH2D1A (Neet and Hunter, 1995; Noguchi November 2003 et al., 1999; Sylla et al., 2000; Sattler et al., 2001). Dok1 mutation in B-CLL S Lee et al 2288 Dok1 downregulates several cell signaling processes: Results for example, it inhibits MAP kinase activity, down- regulates cell proliferation, affects T- and B-cell signal- Frameshift mutation in the coding region of Dok1 ing and plays a role in activin-induced apoptosis in B-CLL (Tamir et al., 2000; Yamanashi et al., 2000; Di Cristofano et al., 2001; Martelli et al., 2001; Nemorin cDNAs isolated from lymphocytes of 11 healthy et al., 2001; Songyang et al., 2001; Wick et al., 2001; individuals and from tumor samples of 46 B-CLL Zhao et al., 2001; Kato et al., 2002; Yamakawa et al., patients were investigated for the presence of mutations 2002). Dok1 can also affect cell adhesion, spreading and in the Dok1 gene. The entire coding sequence was migration (Noguchi et al., 1999; Hosooka et al., 2001). amplified using the primers listed in Table 1 and the Tyrosine phosphorylation and PH domain-mediated PCR products were subjected to heteroduplex analysis. plasma membrane translocation are necessary for Dok1 No abnormality in migration was observed for any PCR function (Liang et al., 2002; Zhao et al., 2001). Dok1 fragment except for one obtained with F4/R4 primers can suppress cell transformation and leukemogenesis in (see Table 1). With this set of primers, a different mice (Di Cristofano et al., 2001; Wick et al., 2001). electrophoretic mobility was seen for B-CLL patient Dok1 maps to human chromosome 2p13 (Nelms et al., number 13, where the fragment migrated faster than in 1998), a region of chromosomal translocation associated the other samples (Figure 1a, lane 3 and data not with several forms of leukemia including B-CLL and B- shown). Sequence analysis of this fragment revealed a cell acute lymphoblastic leukemia (B-ALL) (Sonnier four-nucleotide GGCC deletion at position 683 in exon et al., 1983; Yoffe et al., 1990; Inaba et al., 1991; 5 (amino acid 221) of the coding sequence of Dok1 Richardson et al., 1992; Nelms et al., 1998). Thus, the (GenBank Dok1 cDNA Accession No. NM-001381) function of Dok1 is likely to be altered in these (Figure 1b). This mutation was confirmed by amplifying malignancies. and sequencing a longer cDNA fragment from CLL13 In the present study, we examined whether Dok1 spanning nucleotide 683 (data not shown). The GGCC is mutated in 46 B-CLL cases. We found a deletion was not detected in the nucleotide sequence of four-nucleotide GGCC deletion in the coding region cDNAs from normal individuals (Figure 1b, and data of the gene in leukemia cells from one patient. not shown). The mutation generates a frameshift and This mutation causes a frameshift leading to protein premature termination of the encoded Dok1 protein, truncation at the carboxyl-terminus, with acquisition of with the generation of a hybrid truncated Dok1 protein a novel amino-acid sequence. In contrast to the with 298 amino acids and a theoretical molecular mass wild-type Dok1 protein that has membrane/cytoplasmic of 33 kDa (Figure 1b, c). This predicted mutant protein, localization, the mutant Dok1 accumulates in the referred to as Dok1-CLL13, shares a 220 amino-acid nucleus and contains a functional nuclear localization sequence with wild-type Dok1, comprising the PH signal (NLS). Whereas the wild-type Dok1 down- domain at the N-terminus and part of the PTB domain regulates PDGF-induced MAP kinase activation, the in the middle. However, as a result of the frameshift, mutated Dok1 in CLL has no, or only a slightly Dok1-CLL13 contains a unique 78 amino-acid sequence increased effect on MAP kinase activity. These results at the C-terminal region that is not present in wild-type imply that Dok1 may play a role in the development of Dok1 (Figure 1c, d). Notably, the new sequence, which B-CLL. has no significant homology with any protein in the Table 1 Primers and PCR conditions for amplification of the Dok1 gene Primer Sequences Nucleotide positiona Exon Annealing T (1C) Expected size (bp) F1A 50-CATGGACGGAGCAGTGATG-30 22–40 1, 2 60 361 R1C 50-AAAGGCGTTTCGGCACAG-30 362–379 F2 50-CCTTCCGCCTGGACACTG-30 285–302 2, 3 60 329 R3A 50-TGGCTCCAGTATCTGACTCT-30 593–613 F4 50-GCCTGCATGGCTCCTACG-30 528–545 4, 5 62 290 R4 50-GAGAACATCGTGCCCCTGT-30 798–817 F5 50-ACATCTTCCAGGCAGTTGAGA-30 741–761 5 67 317 R5 50-CTGCGCATGCTCATACAAGT-30 1038–1057 F6 50-GGAGAGGGAGTACAACGGAAG-30 1001–1021 5 60 370 R6 50-TGTACAGGGCTGAGTTGTG-30 1352–1370 F7 50-AGCCTTCCCTGAACCTGGTA-30 1306–1325 5 60 351 R7 50-CATCTGCCCCACTTCTTAGG-30 1637–1656 aDok1 cDNA sequence (GenBank Accession No. NM-001381) Oncogene Dok1 mutation in B-CLL S Lee et al 2289 Cloning and expression of Dok1-CLL13 In order to obtain insight into the possible function of the mutant Dok1 protein, a cDNA fragment amplified from CLL13 using F1A/R5 primers (see Table 1) was cloned in a TA plasmid vector (see Materials and methods).