Evi27 Encodes a Novel Membrane Protein with Homology to the IL17 Receptor

Erming Tian1, Jerey R Sawyer1, David A Largaespada2, Nancy A Jenkins3, Neal G Copeland3 and John D Shaughnessy Jr*,1

1Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas, AR 72205, USA; 2Department of Genetics, Cell Biology and Development, University of Minnesota Cancer Center, Minneapolis, Minnesota, MN 55455, USA; 3Mouse Cancer Genetics Program, National Cancer Institute-FCRDC, Frederick, Maryland, MD 21702, USA

Evi27 is a common site of retroviral integration in BXH2 (Mrvi1); a gene encoding a hematopoietic cell growth murine myeloid leukemias. Here we show that integra- and dierentiation factor, myeloblastosis oncogene tion at Evi27 occurs in a CpG island *6 kb upstream (Myb); three homeobox genes, homeobox A7 (Hoxa7), from a novel gene (designated Evi27) with homology to homeobox A9 (Hoxa9), and myeloid ecotropic viral the IL17 receptor (Il17r) and that proviral integrations integration site 1 (Meis1); a zinc-®nger protein (Evi1); result in increased expression of the Evi27 protein on the and a gene with homology to the ubiquitin-speci®c cell surface. The human EVI27 homolog was also cloned protease 8 (Usp8) oncogene and to genes encoding and mapped to chromosome 3p21. Multiple Evi27 various cell cycle regulatory proteins, ecotropic viral isoforms were detected at the RNA and protein level in integration site 5 (Evi5) (Buchberg et al., 1990; both human and mouse, indicating that Evi27 expression Viskochil et al., 1990; Shaughnessy et al., 1999; is complex. Some of the isoforms are shown to likely Copeland and Jenkins, 1999; Nakamura et al., 1996a; represent secreted soluble forms of the protein produced Morishita et al., 1988; Liao et al., 1997). Four of the by intron incorporation or by proteolytic cleavage. In the genes are proven or suspected human disease genes: mouse, highest Evi27 expression occurs in liver and testes EVI1, NF1 and HOXA9 are causally associated with with lower expression in kidney and lung. In humans, myeloid leukemia and EVI5 with stage 4S neuroblas- EVI27 is expressed at high levels in the kidney, with toma (Ogawa et al., 1996; Copeland and Jenkins, 1999; moderate levels in the liver, brain, and testes. Within Nakamura et al., 1996b; Roberts et al., 1998), hematopoietic cells, Evi27 expression is restricted. validating the usefulness of this approach for human Northern and Western analysis showed that Evi27 is disease gene identi®cation. expressed in selected T-cell, B-cell and myeloid cell lines. Although proviral tagging has identi®ed many These results suggest that Evi27 expression is tightly disease genes, it is apparent that several more genes regulated during hematopoietic dierentiation. Collec- remain to be cloned. This is suggested by the fact only tively, these studies identify a new member of the 45% of BXH2 leukemias contain a virally induced cytokine receptor family whose increased and uncoordi- mutation in one of the genes identi®ed so far. Disease nated expression may lead to myeloid leukemia by genes for 55% of BXH2 leukemias remain to be altering Evi27's normal ability to control the growth identi®ed. The same is true for human acute myeloid and/or dierentiation of hematopoietic cells. Oncogene leukemias (AMLs) where the 11 dierent chromosomal (2000) 19, 2098 ± 2109. translocations and inversions cloned to date are found in only 45% of AMLs (Look, 1997). Disease genes for Keywords: insertional mutagenesis; novel cytokine 55% of AMLs remain to be identi®ed. Ultimately, it receptor; IL-17R related gene; myeloid leukemia should be possible to use proviral tagging to do a saturation screen for BXH2 disease genes. The expectation is that some of these genes will represent Introduction human AML genes that are not easy to clone because they are infrequently involved in human disease or are Retroviral insertional mutagenesis in BXH2 and not marked by a cytologically detectable rearrange- AKXD recombinant inbred (RI) mice induces a high ment. Given the large number of genes that may incidence of myeloid leukemia and the proviral remain to be identi®ed, this task could be dicult using integration sites in the leukemias provide powerful conventional proviral tagging approaches, which rely genetic tags for disease gene identi®cation (Bedigian et on cloning leukemia-speci®c integration sites into al., 1984; Gilbert et al., 1993). During the past several bacteriophage lambda. years, a number of disease genes have been identi®ed in With this potential problem in mind, an inverse PCR these leukemias by proviral tagging. They include a (IPCR) method for proviral tagging was developed that tumor suppressor gene, neuro®bromatosis type 1 (Nf1); makes use of automated DNA sequencing and the a gene with homology to the lymphoid-restricted type genetic tools provided by the Mouse Genome Project, II membrane protein Jaw1, Mrv integration site 1 which greatly increases the throughput of proviral tagging for disease gene identi®cation. More than 400 proviral integration sites from BXH2 myeloid leuke- *Correspondence: JD Shaughnessy Jr mias (and AKXD T- and B-cell leukemias) were cloned Received 10 January 2000; revised 10 March 2000; accepted 14 and characterized using this IPCR method (Li et al., March 2000 1999), which lead to the identi®cation of more than 90 Novel cytokine receptor in myeloid leukemia E Tian et al 2099 new candidate leukemia disease genes (Li et al., 1999). derived from the mouse choline dehydrogenase (Chdh) Nineteen new common integration sites (sites that are gene, while one exon was derived from a gene with targets of viral integration in more than one leukemia) high homology to the yeast actin-like protein, ARP8 were also identi®ed in these studies and BLAST search (accession number S67026). The Chdh gene was and/or chromosome mapping identi®ed candidate subsequently positioned *10 kb to the left of the disease genes for 12 of these common sites (Li et al., proviral integration sites and in the opposite transcrip- 1999). tional direction (Figure 1). Northern analysis showed One common integration site identi®ed by the IPCR that Chdh expression is restricted to liver and is not is Evi27 (Li et al., 1999). While BLAST searches did activated in the B160 cell line (a cell line derived from not identify a candidate disease gene for Evi27, the N57 leukemia that carries a proviral integration at chromosome mapping studies showed that Evi27 maps Evi27 (data not shown). The ARP8 homolog was to mouse chromosome 14 in a region of human 3p21 mapped *15 ± 20 kb to the right of the proviral homology (Copeland et al., 1993). This result is integration sites. Northern analysis showed that this interesting because treatment-related 3p21 breaks are gene is ubiquitously expressed and is not upregulated often observed in myelodysplastic syndrome (MDS) in B150 leukemia cells (data not shown). On this basis, and AML patients (Shi et al., 1996) and 3p21 is the Chdh and ARP8 were excluded as candidate disease most frequently deleted region seen in chronic myeloid genes. leukemia (CML) (Johansson et al., 1997). Further The fourth exon (p57ET47) showed no signi®cant characterization of Evi27 may therefore identify an homology to any sequences in GeneBank. Hybridiza- important human disease gene. In studies described tion studies showed that this exon was contained here, we have identi®ed a novel cytokine receptor- within lambda clone p57l129 (Figure 1), indicating related gene whose expression is upregulated by viral that it was located within 5 ± 20 kb of the proviral integration at Evi27. Future studies are aimed at integration sites at Evi27. DNA sequence analysis of determining whether this is a human disease gene. p57l129 identi®ed a 103 bp fragment that was homologous to a human fetal liver cDNA EST (accession number T96740). This EST sequence was localized 1240 bp upstream of p57ED47 and was found Results to be oriented in the same transcriptional direction, suggesting that both exons might be derived from the Cloning and characterization of genomic sequences from same gene. the Evi27 locus Northern blot analysis of mouse tissues using both Genomic restriction analysis of DNA from two BXH2 exons as probes con®rmed this prediction and showed leukemias with proviral integrations at Evi27 (N57 that the Evi27 hybridization pattern is complex. Six [B160] and 15-38374) showed that the integrations in dierent Evi27 transcripts of approximately 4.4, 4.2, the two leukemias are located about 1.5 kb apart and 2.3, 1.9, 1.3, and 1.1 kb in size, could be detected on are oriented in the same transcriptional direction Northern blots (Figure 2). In adult tissues the (Figure 1). Lambda and P1 clones from the region expression was seen in liver and testes where the 2.3 were then isolated, restriction mapped and exon and 1.9 kb transcripts predominated. Expression of the trapped. Four exon-trapped products were identi®ed. 4.2 transcript was also seen in liver and testes. In BLAST searches showed that two of the exons were addition, low levels of the 2.3 and 1.9 kb transcripts

Figure 1 A partial long-range restriction map of the Evi27 locus. Bacteriophage l and P1 clones used for gene localization and exon trapping are shown above the map. The location of the 5' end and transcriptional orientation of the Evi27 and Cdhd genes are noted by arrows above the map. The location of the exon with homology to the yeast ARP8 gene is also noted. The transcriptional orientation of this gene has not been determined. The position and transcriptional orientation of the proviruses in the B160 and 15- 38374 leukemias are indicated below the map. The cluster of rare cutting restriction enzymes surrounding the proviral integration sites is also noted. The 14 kb BamHI fragment cloned from leukemia B160 is presented below the map. The viral sequences and viral long terminal repeat (LTR) is noted by a thick line and black box, respectively. The position of the 5' end of Evi27 with respect to the viral LTR is noted. Restriction enzymes: B, BamHI; Bs, BssHII; C, ClaI;E,EagI; MluI, N, NaeI; Nr, NruI, S, SacII; Sa, SalI

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2100 were seen in kidney along (Figure 2a). With long which contains a viral integration at Evi27. In this cell exposure, the lung showed expression of a 2.1 kb line the 4.4 kb transcript predominated. These results transcript, while the heart showed expression of the suggest that viral integration at Evi27 results in 1.3 kb transcript. No expression was seen in skeletal increased Evi27 expression. muscle, brain, or spleen. In humans, two EVI27 transcripts, 1.9 and 2.7 kb in In addition to adult tissues, Evi27 expression was size, were detected (Figure 3). Human EVI27 expres- also detected in some hematopoietic cell lines. These sion is therefore considerably less complex than in cell lines include the EL-4 T-cell line, which express the mouse. The highest EVI27 expression was seen in 4.2 and 1.3 kb transcripts, the WEHI231 B-cell line kidney (Figure 3a), while in the mouse, Evi27 was which expresses the 4.2, 1.3 and 1.1 kb transcripts, and expressed at low levels in the kidney. Moderate the R1.1 T-cell line, which expresses the 1.9 kb expression was also observed in the brain, liver, and transcript. In contrast, little or no expression was testes with low to undetectable expression in lung. No detected in the 32D, WEHI 3B, and M1 myeloid cell EVI27 expression was seen in heart, placenta, and lines, the HYB.548 B-cell line, or the P815 mast cell skeletal muscle (Figure 3a) or in immune tissues such line (Figure 2c). as spleen, lymph nodes, thymus, peripheral blood Evi27 expression could also be detected in BXH2 lymphocytes, or bone marrow (Figure 3b). High myeloid cell lines whether or not they carry a proviral EVI27 expression was, however, observed in the fetal integration at Evi27. These cell lines expressed. liver. Expression of the 4.4, 4.2, 1.9 and 1.1 kb transcripts Within cell lines, moderate EVI27 expression was was variable among the dierent lines (Figure 2c), but seen in the cervical carcinoma HeLa, chronic myelo- the highest expression was seen in the B160 cell line, genous leukemia, K562, and colon adenocarcinoma,

Figure 2 Northern blot analyses of Evi27 expression in murine tissues and cancer cell lines. Multiple tissue Northern blots of normal adult (a), BXH2 leukemic cell lines (b), and various hematopoietic cell lines (c): M1, myeloid leukemia; WEHI3B, monocyte; HYB. 548, B-cell hybridoma; WEHI231, preB-cell lymphoma; R1.1, lymphocytic thymoma; EL4, thymoma, P815, mastocytoma. The blots were also hybridized with a GAPDH or b-actin probe to control for RNA loading. The size of kilobases (kb) of molecular weight markers is shown on the left of each panel. To the right of each panel are the sizes of the Evi27 transcripts observed

Figure 3 Northern blot analyses of EVI27 expression in human tissues and cancer cell lines. Multiple tissue Northern blots of normal adult (a), immune tissues (b) and cancer cell lines (c) hybridized with an EST speci®c for the human EVI27 gene. The human cancer cell lines are HL60, promyelocytic leukemia; HeLa, cervical carcinoma; K562, chronic myelogenous leukemia; MOLT4, T- lymphoblastic leukemia; Raji, Burkitt's lymphoma; SW480, colon adenocarcinoma; A549, lung carcinoma; G361, melanoma. The blots were also hybridized with a b-actin probe to control for RNA loading. The size in kb of molecular weight markers is shown on the left of each panel

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2101 SW480 cell lines (Figure 3c) but not in the myeloid and ends at residue 309. The cytoplasmic tail is leukemia HL-60, acute lymphoblastic T-cell leukemia, predicted to extend from residue 310 to the C terminus. MOLT-4, Burkitt's lymphoma, Raji, lung carcinoma, pSORT analysis also indicated that the protein has A549 and the melanoma, G361 cell lines. type 1a topology. A string of 39 aa derived from intronic sequences would be inserted at amino acid 250 in the 2.7 kb transcript. This would result in the The gene whose expression is upregulated by viral synthesis of a 288 aa protein with a predicted integration at Evi27 encodes a novel IL17 receptor-related molecular weight of 31 kD. The transmembrane and protein cytoplasmic portions of the 502 aa isoform are missing The complete coding region of human gene was in this truncated form of the protein. obtained by 3', and then 5' RACE. The primary 5' Multiple 5' RACE products were also obtained in RACE product using primers from the 3' end of the mouse. Cloning and sequence analysis of these RACE gene was *1.9 kb in length, consistent with the size of products showed that they can be created by the the major human transcript. A larger minor species inclusion of at least two unspliced introns. As in was also produced in the 5' RACE reaction. Sequence human, these intron sequences introduce multiple stop analysis showed that it contained an additional 950 bp codons into the open reading frame of the protein. The of sequence that was not present in the 1.9 kb product. sequence of the full length 1.9 and 2.5 kb mouse PCR and genomic sequence analysis showed that this transcripts has been deposited in GenBank (accession extra sequence was an unspliced intron. Northern numbers AF208108 and AF208109, respectively). The analysis showed that the 2.7 kb human transcript mouse 4.4, 4.2, 2.4 and 1.3 kb transcripts have not contains this unspliced intron (data not shown) and been cloned and sequenced. Genomic sequence analysis sequence analysis showed that this unspliced intron indicates that the mouse gene consists of at least 13 introduces multiple stop codons into the open reading exons and spans *15 kb of genomic DNA. The gene is frame of the protein. transcribed in the same orientation as the integrated The sequence of the full length 1.9 and 2.7 kb proviruses, which lie in a CpG rich region located transcripts has been deposited in GenBank (accession approximately 6 kb 5' from the ®rst known exon of the numbers AF208110 and AF208111, respectively). The gene (Figure 1). The predicted open reading frame of predicted uninterrupted protein is 502 amino acids (aa) the 1.9 kb transcript is 499 aa with a predicted and has a predicted molecular weight of 56 kD (Figure molecular weight of 56 kD (Figure 4). pSORT analysis 4). pSORT analysis identi®ed a potential cleavable identi®ed a potential cleavable signal peptide in the N signal peptide at the N terminus of the protein. A terminus of the protein, just like in the human protein. putative transmembrane domain starts at residue 293 A putative transmembrane domain starts at aa 290 and

Figure 4 Alignment of mouse and human Evi27 proteins. The putative signal peptide, transmembrane domain, and peptide used to generate polyclonal antisera are indicated by a black line over the sequence. Conserved protein motifs are indicated by a double line. Conserved amino acids are boxed with amino acid identities noted in bold. Normal typeface letters represents conservative amino acid changes. Gaps created in the sequences to optimize alignments are indicated by a dash in the sequence string. Abbreviations: N- GLY, N-linked glycosylation; GSK, kinase phosphorylation site

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2102 ends at aa 293. The transmembrane domain is located The full-length human and mouse proteins are 83% in exon 12 of the gene and is spliced out in variant similar and 76% identical (Figure 4). The extracellular transcripts sequenced from the WEHI-231 B-cell line. domain of both proteins contains three conserved N- These variant transcripts splice out an exon containing linked glycosylation and a GSK3 phosphorylation site. the entire putative transmembrane domain. The lack of A single conserved GSK3 phosphorylation site is also this exon does not disrupt the predicted open reading present in the cytoplasmic domain. frame of the protein. Amino acid sequence comparisons showed that In the 2.5 kb transcript, a string of 57 novel amino Evi27 has signi®cant homology throughout its coding acids, derived from intron 7, is added at aa 162 region to the human and mouse IL17 receptor (IL17R) creating a protein with an open reading frame of (E value=2.5 e-29) (Figure 5). The position of the 218 aa and a predicted molecular weight of 24 kD. transmembrane domain with respect to the amino Like the shorter human isoform, this isoform would terminus is essentially the same in the two proteins. lack the transmembrane and cytoplasmic domains of However, the cytoplasmic tail of the IL17R is nearly the protein. 304 aa longer than that of the Evi27. Although Evi27 is

Figure 5 Alignment of the human EVI27 and IL17R proteins. Conserved amino acids are boxed with amino acid identities noted in bold. Gaps created in the sequences to optimize alignments are represented by dashes. Amino acid positions are indicated to the right and left of the sequence

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2103 predicted to encode both membrane bound and soluble length protein, the transfected line expresses approxi- forms, IL17R is not known to encode a soluble form. mately 3 ± 5-fold higher levels of this isoform (data not shown). Immuno¯uorescence analysis showed weak staining in the parental line whereas transfected cells The mouse and human genes map in syntenic regions showed more abundant cytoplasmic staining (Figure The human EVI27 gene was mapped to chromosome 7a). Clear cell surface staining was observed after 3p21 by ¯uorescence in situ hybridization of high- analysis of live cells or ®xed, but not permeabilized, resolution G-banded chromosomes (Figure 6). These transfected cells (data not shown). In all experiments results are consistent with the mouse mapping data, the staining could be quenched by preincubation of the which localize the mouse gene to chromosome 14 in a antisera with Evi27 peptide (Figure 7a, right panels). region of human 3p21 homology (Copeland et al., In contrast to the low level expression in the 32D 1995). They also con®rm that the human gene maps in myeloid leukemia line high level of both cytoplasmic a region of the human genome that is frequently and cell surface expression of the protein was noted in rearranged in human myeloid leukemia. the cell line B160 (Figure 8). Although several other BXH2 cell lines also showed expression, the highest expression was seen on B160 cells, consistent with Expression and subcellular localization of Evi27 Northern results showing that Evi27 mRNA is over- The expression and subcellular localization of the expressed in B160 cells (Figure 2). mouse protein was examined using a polyclonal antibody raised against a conserved peptide derived Western blot analysis of Evi27 protein expression from the extracellular domain of the N-terminus of Evi27 (Figure 4). Antibody speci®city was con®rmed Western blot analysis of a number of murine leukemia by Western blot and immuno¯uorescence staining of cell lines con®rmed the Northern results and showed that 32D cells, which express undetectable levels of Evi27 many dierent Evi27 isoforms are expressed at the mRNA (Figure 7b) and 32D cells stably transfected protein level (Figure 9). A 56 kD protein, which is the with a full length 1.9 kb Evi27 cDNA clone. Western predicted size of the full length protein containing the blot analysis showed that whereas the parental 32D transmembrane and cytoplasmic domains, was expressed cells show weak expression of the expected 55 kD full in most samples tested (Figure 9a). The EL4 and R1.1 T- cell lines and the WEHI 231 B-cell line also expresses a 24 kD protein, which is the predicted size of the truncated protein resulting from intron incorporation and an 85 kD protein (Figure 9a). This protein is also expressed at low levels in the B190 cell line but not in the other BXH2 leukemia cell lines tested. Several cytokine receptors also produce soluble forms of the receptor by proteolytic cleavage of the membrane bound receptor. The cleavage sites are located near the plasma membrane in the extracellular domain of the receptor. Proteolytic cleavage of the Evi27 membrane receptor in this region is predicted to produce a 33 kD protein. A band of this size is also present in most samples tested (Figure 9a). Several unexpected sized Evi27 bands were also detected on the Western blot. These protein bands can all be competed away by the addition of Evi27 peptide (Figure 9b), indicating that they contain authentic Evi27 protein sequences. The origin of these protein bands is unknown but could result from the translation of Evi27 mRNA transcripts that have yet to be sequenced or may be indicative of homo- or heteropolymer formation. The expression of these protein bands is sometimes variable. For example, the 75 kD band is expressed at low or undetectable levels in the B160 and B187 cell lines. The B193 cell line does not express the 150 and 127 kD bands, but does express high levels of the 75 and 33 kD bands. The reason for this is not clear, but may re¯ect the dierentiation state of the cell line. Consistent with this hypothesis, T- and B-cell lines express Evi27 protein bands that are not expressed in BXH2 myeloid cell lines (i.e., 24 and 85 kD bands).

Figure 6 Chromosome mapping of the human EVI27 gene. (a) Fluorescence in situ hybridization of a PAC clone speci®c for Discussion EVI27 gene to normal human metaphase chromosomes. Arrows indicate localization of the probe. (b) G-banding of the same metaphase spread in (a) following destaining of FISH reagents. In studies described here we show that retroviral The probe is localized to band 3p21 integration at the Evi27 locus in BXH2 murine myeloid

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2104

Figure 7 Immuno¯uorescence and Northern blot analysis of Evi27 expression in murine cell lines. (a) Immuno¯uorescence staining of the Evi27 protein (red) in the myeloid cell line 32D (top panel) and 32D cells transfected with an Evi27 cDNA expression construct (lower panel). Note the light perinuclear staining in the parental line and abundant staining in the transfectant. Nuclei are stained blue with DAPI. Cells were also stained with Evi27 antibody preincubated with Evi27 peptide. Note reduced red staining is evident, demonstrating speci®city of the antisera. (b) Northern blot hybridization of poly(A)+ mRNA from 32D and 32D/Evi27A stable transfectants. Note the abundant expression of the transgene in the transfectant and the absence of expression in the parental line. The blot was also hybridized with a b-actin probe to control for RNA loading. The size in kb of molecular weight markers is shown on the left of each panel

Figure 8 Immuno¯uorescence staining of Evi27 protein in BXH2 leukemia cell line B160. Fixed and permeabilized cells (a) or live cells (b) stained with anity puri®ed rabbit anti-Evi27 antibody followed by rodamine (a) or FITC (b) conjugated goat anti-rabbit IgG (left panels). Cells were also stained with Evi27 antibody preincubated with Evi27 peptide (a) or normal rabbit IgG (b). Nuclei are stained blue with DAPI

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2105

Figure 9 Western blot analysis of Evi27 protein expression in murine and human cell lines. (a) BXH2 leukemia cell lines, EL4 and R1.1 T-cell lines, and the WEHI231 B-cell line. Ten mg of protein from cell lysates was run in tandem and hybridized with anity puri®ed Evi27 antibody either without (a) or with (b) preincubation with Evi27 peptide. Note the absence or reduction of speci®c bands in panel (b) compared to (a). Molecular weight standards in kD are indicated to the left. The Evi27 isoforms and sizes are indicated by arrows to the right of a leukemias result in the increased expression of a novel membrane bound forms and it is now well documented gene with homology to the IL17 receptor. A number of that the soluble receptors can have both positive and cytokine receptors (i.e., v-mpl or Tpor, Il6ra, Epor, Il9r, negative eects on ligand signaling (for review see Csf1, Il2ra, Il4ra and Ifngr2) or their ligands (i.e., Il6, Heaney and Golde, 1996). The origin of the other Csf1, Il3) are reported targets of retroviral integration Evi27 transcripts has not been determined. It is thus or transduction in mouse leukemia/lymphomas making possible that one or more of these uncharacterized cytokines/cytokine receptors one of the largest and messages encode sequences that are located upstream most important classes of leukemia genes (Ymer et al., of Evi27 proviral integration sites at Evi27. If this is the 1985; Penciolelli et al., 1987; Gisselbrecht et al., 1987; case, then Evi27 proviral integrations may prevent Baumbach et al., 1988; Sugita et al., 1990; Blankenstein these messages from being expressed and this may have et al., 1990; Souyri et al., 1990; Hino et al., 1991; important disease consequences. Chretian et al., 1994; Flubacher et al., 1994; Li et al., Il17r/Evi27 receptors are unusual in that they have 1999). The Evi27-encoded cytokine-related receptor is no homology with any protein in public databases, therefore an excellent candidate for a new leukemia including other cytokine receptor proteins, and they disease gene. have no recognizable motifs associated with intracel- The human EVI27 gene was also cloned and mapped lular signaling (Yao et al., 1995b, data presented here). to chromosome 3p14-p21, in a region syntenic to the Many disease-related cytokine receptors involved in mouse gene. The human protein is 76% identical to the leukemogenesis contain kinase domains, however, it is mouse protein at the amino acid level. This is not unprecedented to ®nd receptors lacking kinase somewhat higher than the recently described homology domains that are involved in disease. For example, the between the mouse and human IL17 receptors, which human MAS1 oncogene, which encodes a functional are 69% identical at the amino acid level (Yao et al., angiotensin receptor, is a G-protein coupled receptor 1997). This conservation suggests that Evi27 receptor that functions through phosphatidylinositol 4,5-bispho- function is evolutionarily conserved between human sphate hydrolysis (Young et al., 1986; Jackson et al., and mouse. 1988). Evi27 may thus identify a new disease pathway. Proviral integrations at Evi27 are located about 6 kb At least seven dierent Evi27 isoforms (i.e., 24, 33, upstream from the ®rst known exon of the IL17 56, 47, 75, 127 and 150 kD in size) were detected in receptor-related gene and result in increased gene BXH2 leukemia cell extracts by Western analysis using expression. Viral integration at Evi27 may thus induce an Evi27-speci®c antibody. All leukemia extracts disease simply by upregulating or constitutively expressed the 56 kD isoform (the membrane bound activating receptor expression. However, given the receptor) as well as the 33 kD isoform (a postulated complex expression pattern observed for Evi27,itis proteolytic cleavage containing the extracellular ligand- also possible the viral integration induces disease by binding domain of the receptor). Expression of the altering Evi27 isoform expression. In the mouse, as other isoforms was variable among the extracts. One of many as six Evi27 transcripts are detected on Northern seven cell extracts (B190) expressed the 24 kD isoform blots. Two of the transcripts result from intron (a putative truncated receptor resulting from intron incorporation and are predicted to produce a truncated incorporation), three extracts (B139, B160 and B187) soluble form of the receptor lacking the transmem- express the 47 kD isoform, ®ve extracts (B112, B132, brane and cytoplasmic domains. Several cytokine B139, B190, B193) the 75 kD isoform, and six extracts receptors are known to produce soluble as well as (B112, B132, B139, B160, B187, B190) the 150 kD

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2106 isoform. The origin of these larger isoforms (i.e., but dramatic granulopoiesis in vivo, except in IL6- oligomerization) or the reason for their variable de®cient mice (Schwarzenberger et al., 1998). expression (i.e. dierentiation state of the cell) is The Evi27 protein is a member of the IL-17 receptor presently unclear. Western blot analysis proteins family. Two new members of the IL-17 cytokine family derived from human cell lines shows that only two have recently been identi®ed (Li et al., 2000). These isoforms (55 and 29 kD) are produced (data not studies have demonstrated that IL-17B and IL-17C do shown). not bind the IL-17 receptor extracellular domain, Only one BXH2 leukemia cell extract, B160, indicating that IL-17B and IL-17C are likely to bind analysed on Western blots is known to contain a unique, yet uncharacterized receptors (Li et al., 2000). proviral integration at Evi27. The 56 kD isoform is In a survey of cytokine induction, IL-17B and IL-17C overexpressed in B160 cells, consistent with Northern stimulate the release of TNFa and IL-1b from the results showing that viral integration at Evi27 results in monocytic leukemia cell line THP-1 (Li et al., 2000). increased Evi27 expression. Surprisingly, the 56 kD RT ± PCR analysis has shown that IL-17B is expressed isoform is also overexpressed in B187 cells, which are by both the B160 leukemia and the stromal cell on not known to carry a viral integration at Evi27. which it depends (JS, unpublished data). We speculate Perhaps, B187 cells harbor a viral integration at that Evi27 may represent the receptor for IL-17B or Evi27 that maps outside the region examined on IL-17C. Based on these data a model for Evi27 in Southern blots or there are other mechanisms for myeloid leukemia development is proposed. Terminal upregulating expression of this isoform other than dierentiation of myelomonocytic precursor cells likely proviral integration. It is interesting to note that B187 result in the down regulation of Evi27 expression. cells, like B160 cells, also fail to express the 75 kD However, proviral insertions at Evi27 result in isoform. One intriguing possibility is that viral constitutive expression of the receptor. Binding of IL- integration at Evi27 blocks expression of this isoform. 17B/C to the Evi27 receptor would trigger the release Evi27 expression is restricted in the hematopoietic of TNFa and IL-1b by the leukemic cell. The TNFa cells. Northern analysis showed that Evi27 is expressed and IL-1b would in turn provoke the production of in T cells and ESTs homologous to the mouse gene multilinage hematopoietic growth factors, adhesion have been identi®ed in CD4+ T-cells. Likewise, the molecules, and in¯ammatory cytokines by stromal cells mouse preB-cell line WEHI231 expresses Evi27 and (Bagby, 1994). These stromal cell derived factors then ESTs homologous to EVI27 have been identi®ed in support the growth and survival of the leukemia cell human germinal center B cells and follicular lympho- and may account for the absolute dependence of the mas. No Evi27 expression was seen in the late stage B- B160 leukemia on the stromal feeder layer for growth cell line 548, the Burkitt lymphoma cell line Raji, the and survival. murine erythroid line D1b, or the mast cell line P815. The human homolog of Evi27 may also be involved A complex pattern of expression was observed within in human disease. EVI27 maps to chromosome 3p21, a the myeloid compartment. Variable expression was region consistently deleted in a variety of human seen in BXH2 leukemia cells, while little or no Evi27 cancers. Loss of 3p heterozygosity is also frequently expression was seen in the M1 and 32D murine observed in renal cell carcinoma, lung cancer and myeloid leukemia cell lines or in the monocytic breast cancer and analysis of 3p allele loss in renal cell leukemia cell line WEHI-3B. Evi27 is expressed, cancer has localized a candidate tumor suppressor gene however, in the chronic human myelogenous leukemia to 3p21 (van den Berg et al., 1997). These results are cell line K562, but not in the promyelocytic leukemia consistent with the hypothesis that chromosome 3p cell line HL-60. These results suggest that Evi27 encodes a number of tumor suppressor genes. Given expression may be tightly regulated during myeloid that EVI27 maps to 3p21 and is expressed at high cell dierentiation and imply that Evi27 may have an levels in normal human kidney, it will be interesting to important function in controlling the growth and/or determine whether this gene is aected by 3p21 dierentiation of hematopoietic cells. Proviral integra- mutations in renal cell carcinoma. EVI27 also may be tion at Evi27 may interfere with this function(s) and, in a human myeloid leukemia disease gene. Recurrent doing so, lead to myeloid disease. treatment-related chromosome 3p21 breaks are fre- IL17 (CTLA8) is a homodimeric cytokine of about quently observed in myelodysplastic syndrome and 32 kD expressed exclusively from human memory T acute myeloid leukemia patients (Shi et al., 1996) and cells or mouse alpha beta TCR+CD47CD87 thymo- 3p21 is the most frequently deleted region in human cytes (Rouvier et al., 1993; Yao et al., 1995a,b; CML (Johansson et al., 1997). Future studies will be Kennedy et al., 1996). In contrast with the tightly aimed at determining whether EVI27 is a human controlled expression of the ligand, the IL17 receptor is myeloid leukemia disease gene. ubiquitously distributed but more abundant in spleen and kidney (Yao et al., 1995b). Although devoid of direct eects on cells of hematopoietic origin, IL17 Materials and methods induces the secretion of IL6, IL8, PGE2, MCP1 and G-CSF by adherent cells like ®broblasts, keratinocytes, BXH2 mice and leukemic cell lines epithelial and endothelial cells (Yao et al., 1995a,b; The BXH2 recombinant inbred strain was obtained from the Fossiez et al., 1996). When cultured in the presence of Jackson Laboratory (Bar Harbor, ME, USA) and maintained IL17, ®broblasts can sustain the proliferation of at the NCI-Frederick Cancer Research and Development CD34+ human progenitors and their preferential Center. BXH2 leukemia cell lines have been previously dierentiation into neutrophils (Fossie et al., 1996). described (Largaespada et al., 1995). Other cell lines were Adenovirus-mediated transfer of murine IL17 cDNA purchased from the ATCC and grown in either RPMI 1640 into liver has also been shown to induce a transient, or DMEM supplemented with 2 mM gluatmine, 4.5 g/L

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2107 glucose, Pen-Strep, and 10% fetal bovine serum (Atlanta precipitations. Insert DNA was then ligated to a BamHI Biologicals, Atlanta, GA, USA). digested pSPL3 vector (Life Technologies, Gaithersburg, MD, USA). DNA from the ligations was used to transform DH10B maximum eciency cells (Life Technologies). Addi- DNA extraction and Southern blot hybridization tional steps in the experiment were performed exactly High molecular weight genomic DNAs were extracted from according to the manufacturers protocol (Life Technologies). frozen normal tissues and leukemic spleens and lymph nodes DNA sequence of approximately 100 individual clones from as previously described (Jenkins et al., 1982). Bacteriophage each ligation was generated as described below. and plasmid DNAs were puri®ed using standard procedures (Sambrook et al., 1987). Restriction enzyme digestions, Poly(A)+ RNA isolation and Northern blot analysis agarose gel electrophoresis, Southern blot transfers, hybridi- zations, and washes were performed as previously described Premade Northern blots containing 2 mg of twice selected (Sambrook et al., 1987). poly(A)+ RNA from various normal tissues and cell lines were purchased from commercial source (Clontech, Palo Alto, CA, USA). Total RNA was extracted from cell line Genomic cloning suspensions by the RNAzol B method (Tel-Test, Friends- Seventy mg of DNA from leukemia N57 was digested to wood, TX, USA). Poly(A)+ RNA was puri®ed from the total completion with BamHI and fractionated by electrophoresis RNA preps by oligo-d(T) column chromatography according in TAE buer. Fragments approximating the somatically to the manufacturers recommendations (Amersham Pharma- acquired pAKV5 (ecotropic murine leukemia virus) hybridiz- cia Biotech, Piscataway, NJ, USA). Two to ®ve mgof ing fragments were cut from the gel and puri®ed using Qiaex poly(A)+ RNA was fractionated by electrophoresis in 1.0% beads according to the manufacturer's instructions (Qiagen, agarose gels containing formaldehyde and transferred to Valencia, CA, USA). The BamHI fragments were cloned into Hybond N+ membranes (Amersham Pharmacia Biotech). EMBL4 phage arms (Stratagene, La Jolla, CA, USA). The membranes were prehybridized and hybridized according Ligated material was packaged with Gigapack Gold packa- to the method of Church and Gilbert or using ExpressHyb ging extracts (Stratagene). Library screening was performed solution (Clontech). Blots were then exposed to X-ray ®lm at according to the protocol of Benton and Davis (1977). The 7708C with an intensifying screen. P1 and lambda genomic clones were derived from commercial libraries (Genome Systems, St Louis, MO, USA; Clontech, cDNA cloning Palo Alto, CA, USA). cDNA cloning was carried out by a combination of 5' and 3' rapid ampli®cation of cDNA ends (5' and 3' RACE) and Human chromosome mapping modi®ed RT ± PCR as described (Shaughnessy et al., 1999). Metaphase chromosomes from normal peripheral blood Brie¯y, the nucleotide sequence of the trapped exon samples were prepared using conventional methods. Slides p57P1ET47 was used to design speci®c nested oligonucleo- were incubated in 26SSC (pH 7.0, 378C ) for 15 min then tides. The primary 5' and 3' RACE reactions were performed immersed in 0.1 N HCl/0.05% Triton X-100 for 15 min at using mouse liver Marathon Ready cDNA (Clontech) as a room temperature (RT). Slides were washed in 26SSC template according to the manufacturers protocol using the (pH 7.0) twice at RT, then washed in 16PBS, pH 7.2 once at Advantage cDNA ampli®cation kit (Clontech). After cloning RT. Slides were immersed in 1% formaldehyde (diluted in and sequencing, nested primers speci®c for the 5' and 3' ends PBS) for 10 min at RT, washed twice in 16PBS at RT, then of the gene were synthesized. Another cycle of PCR was once in 26SSC (pH 7.0) at RT. Chromosomes were performed using Marathon Ready liver cDNA as a template dehydrated in an ethanol series and air dried at RT. A and the full length cDNA was generated by PCR as follows: human PAC clone speci®c for EVI27 was isolated according one cycle: 948C, 1 min; 30 cycles: 948C, 30 s, 688C, 7 min. to the manufacturer's instructions (Genome Systems, St The products of the reaction were subcloned into pCR2.1 Louis, MO, USA). Spectrum-Red-dUTP (Vysis, Downer's (Invitrogen, Carlsbad, CA, USA) and sequenced. The full- Grove, IL, USA) was incorporated into the PAC probe by length cDNA of the 1.9 and 2.7 kb transcripts have been nick translation according to the manufacturer's protocol submitted to GenBank under accession numbers AF208108 (Vysis). DNA was resuspended in Hybrisol VII (Oncor, and AF208109, respectively. Human cDNAs covering the Gaithersburg, MD, USA) (50% formamide/26SSC) at a coding region of the EVI27 gene were synthesized in a similar ®nal concentration of 5 ng/ml and hybridized to chromo- manner as described above. Brie¯y, a human EST (accession somes according to manufacturer's instructions. The chromo- number T96740), with high homology to the mouse EVI27 somes and probe solution was incubated at 758C for 10 min cDNA, was used to design nested oligonucleotide primers at then to 378C for 16 h in a humidi®ed chamber. Post the 5' and 3' end of the EST. 5' and 3' RACE products from hybridization washes consisted of 65% formamide/26SSC Marathon Ready fetal liver cDNA (Clontech) were synthe- (pH 7.0) for 15 min at 438C, then 26SSC (pH 7.0) for 8 min sized as described above. The 1.9 and 2.9 kb human EVI27 at 378C,16PBD (Oncor) twice at RT, then in 16PBS cDNA nucleotide sequences are deposited in GenBank under (pH 7.2). Finally, 10 ml of propidium iodide/antifade (1 : 20) accession numbers AF208110 and AF208111. was added on each area and a coverslip added. Following FISH the slide was washed twice in Xylene to remove the Transfection and establishment of stable cell lines coverslip and excess oil. The slide was then washed in 16PBD at 378C for 5 min, followed by a dehyrdration step The 1.9 kb mouse Evi27 cDNA was cloned into the NotI± through an ethanol series. The slide was then incubated in HindIII site of the pcDNA3.1(7) eukaryotic expression 50% formamide/26SSC for 90 min at 378C, washed brie¯y vector and transformed into DH5a bacteria. Ten mgof in tap water, followed by dehydration in 10, 80 and 95% recombinant plasmid DNA was resuspended at 1 mg/mlin ethanol and stained with Wright's stain/phosphate buer for sterile TE. Cell lines 32Dc13, M1, WEHI3B and NIH3T3 5 min. were grown in appropriate media maintaining cell viability at 90%. Cells were harvested and washed 36in FBS-free DMEM media. Cells were resuspended at 56106 cells per Exon trapping ml in FBS-free DMEM. One ml of the cell suspension was The P1 clone p57P1 was digested to completion with BglII mixed with DEAE dextran at 0.1 mg/ml and 10 mg of plasmid and BamHI and puri®ed by phenol extractions and ethanol DNA in an 0.4 cm electrode gap electroporation cuvette and

Oncogene Novel cytokine receptor in myeloid leukemia E Tian et al 2108 chilled on ice for 10 min and Electroporated at 270 V/975 mF primary antibody and the solution was added to slides for in a Gene Pulser II (Bio-Rad, Hurcules, CA, USA). The 30 min. Slides were washed 36in PBS for 5 min each. The cuvette was chilled on ice for 10 min then the cells were nuclei were counterstained with DAPI at 1/40 dilution in transferred into 10 ml of fresh media and grown for 72 h. antifade (Oncor), coverslips added and viewed with an G418 selection was carried out in 2 mg/ml for 2 weeks. Olympus BX60 epi¯uorescence microscope. Limiting dilution cloning in 96 well culture dishes was For live cells all procedures were performed on ice. Cells performed at 1 mg/ml G418 on all cell lines with the were washed twice in cold PBS, incubated with 3%BSA/3% exception of NIH3T3. Stable transfectants were cloned and goat serum for 30 min then stained with either 5 mgof tested for expression of the transgene by Northern blot primary anti-Evi27 or normal rabbit IgG for 15 min, washed hybridization. once in PBS, then stained with a 1 : 20 dilution of FITC conjugated goat anti-rabbit antibody (Vector Laboratories) for 15 min, and washed once and resuspended in cold PBS. Southern and Northern blot probes Cells were placed on slides by cytocentrifugation and were The b-actin probe was a 2.0 kb cDNA (Clontech). Probes allowed to air dry for 15 min at RT. The nuclei were pAKV5 and pEco have been described (Nakamura et al., counterstained with DAPI and visualized as above. 1996a,b). p57BXH2A was a 700 bp PCR fragment generated from the ¯anking DNA from clone p57BXH2. Probe Western blotting p57ET47 was a 103 bp exon trapped from p57 P1. The human intron probe was synthesized by PCR from genomic Cells were harvested at logarithmic growth stage and washed DNA. Human ESTs were derived from IMAGE consortium 26in 1 XPBS. Cells were resuspended in extraction buer clones. Full length 1.9 kb human and mouse Evi27 cDNAs (16PBS (pH 7.2), 10 mg/ml Aprotinin (0.1 unit/ml), 10 mg/ml were used in all probes and were labeled with a32P-dCTP Leupeptin, 1 mM PMSF) at 106 cells/100 ml. Protein was using the Prime It II labeling kit (Stratagene). extracted by the freeze-thaw method. The solid phase was removed by centrifugation at 12 000610 min at 48C. The aqueous phase was collected concentrated and quanti®ed DNA sequencing using the BCA assay kit (Pierce). Protein separating gels were DNA sequencing was performed using the PRISM Ready precast (Novex, San Diego, CA, USA). Protein (50 ± 100 mg) Reaction DyeDeoxy Terminator Cycle Sequencing Kit was mixed 1 : 1 with sample buer (26) (Novex), denatured (Perkin Elmer) on the ABI Model 373A DNA Sequencer for 15 min, cooled to RT and loaded. Gels were run for 2 ± (Applied Biosystems). Sequence primers were either the T3, 3 h at 200 V with cooling (60 W initially) in 16running T7 sequencing primers or synthetic oligomers derived from buer (25 mM Tris, 192 mM glycine, 0.1% SDS at pH 8.3). previously determined sequence. Protein was transferred to Hybond-C super membrane (Amersham Pharmacia Biotech) using an electrophoretic blotting system (CBS Scienti®c Co. Del Mar, CA, USA) in Immunohistochemistry 16transfer buer (48 mM Tris, 39 mM glycine, 20% Cells were washed once in PBS, resuspended in PBS and methanol) at 100 V for 5 ± 6 h. The Western blot was subjected to cytocentrifugation at 12006g for 5 ± 10 min. processed as described (WesternBreeze kit, Novex, San Cells were air dried for 15 ± 20 min, rinsed in PBS and Diego, CA, USA). drained well but not allowed to dry. Cells were ®xed in fresh 4% paraformaldehyde for 10 min followed by two washes in Computer DNA and protein sequence analysis PBS, then permeabilized by incubation in acetone for 30 s at RT. Slides were rinsed in PBS with four changes over 5 min. Sequence homology searches were conducted at the protein Cells were blocked with 5% goat serum 30 min at RT. The level using the National Center for Biotechnology Informa- polyclonal rabbit anti-EVI27 antibody, #2954, was anity tion and the BLAST network service. The DNA and protein puri®ed using the Sulfa-Link kit according to manufacturer's sequence alignments and protein motif sequence searches instructions (Pierce, Rockford, IL, USA). Antibodies were were performed using MacVector (Oxford Molecular Group, diluted in 1 M HEPES buer containing 0.15 N NaCl. Sixty Beaverton, OR, USA) and pSORT (Nakai and Kanehisa, microliters of the primary antibody solution was added to 1992). slides and incubated for 30 min in a humidi®ed chamber at RT. The primary antibody was blocked with 100 mg of Evi27 peptide (Figure 4) by incubating for 1 h at RT. This solution was then added to the slides as above. After hybridization the Acknowledgments slides were washed three times for 5 min in 16PBD (Oncor). We thank Diane E Hasz for technical assistance. This The secondary antibody a rhodamine conjugated goat-anti- research was sponsored in part by the National Cancer rabbit antibody (Pierce) was diluted in the same buer as the Institute, DHHS, under contract with ABL.

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