Oncogene (1999) 18, 2069 ± 2084 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Mrvil, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1 John D Shaughnessy Jr*,1, David A Largaespada2, Erming Tian1, Colin F Fletcher3, Brian C Cho4, Paresh Vyas5, Nancy A Jenkins3 and Neal G Copeland3 1Division of Hematology and Oncology, Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, AR 72205, USA; 2Department of Laboratory Medicine and Pathology, University of Minnesota Cancer Center, Minneapolis, Minnesota, MN 55455, USA; 3Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland, MD 21702, USA; 4Department of Microbiology and Immunology, Thomas Jeerson University Cancer Institute, Philadelphia, Pennsylvania, PA 19107, USA; 5Department of Hematology and Oncology, Children's Hospital, Boston, Massachusetts, USA Ecotropic MuLVs induce myeloid leukemia in BXH2 Introduction mice by insertional mutagenesis of cellular proto- oncogenes or tumor suppressor genes. Disease genes BXH2 mice represent an important model for the can thus be identi®ed by viral tagging as common sites of identi®cation of myeloid leukemia disease genes. Not viral integration in BXH2 leukemias. Previous studies only do these mice have the highest spontaneous showed that a frequent common integration site in BXH2 incidence of myeloid leukemia of any known inbred leukemias is the Nf1 tumor suppressor gene. Unexpect- mouse strain, but the leukemias in these mice are edly, about half of the viral integrations at Nf1 retrovirally-induced and the proviruses in the tumors represented a previously undiscovered defective none- can thus be used as insertional tags to identify the cotropic virus, termed MRV. Because other common disease genes. BXH2 is one of 12 recombinant inbred integration sites in BXH2 leukemias encoding proto- (RI) BXH strains derived from crossing C57BL/6J and oncogenes contain ecotropic rather than MRV viruses, it C3H/HeJ mice. Unlike the parental strains, or the 11 has been speculated that MRV viruses may selectively other BXH R1 strains, BXH2 mice spontaneously target tumor suppressor genes. To determine if this were express a B-ecotropic murine leukemia virus (MuLV) the case, 21 MRV-positive BXH2 leukemias were beginning early in life and 495% of these mice die of screened for new MRV common integration sites. One myeloid leukemia by 1 year of age (Bedigian et al., new site, Mrvi1 was identi®ed that was disrupted by 1981, 1984). The BXH2 B-ecotropic virus is not MRV in two of the leukemias. Ecotropic virus did not transmitted through the germline (Jenkins et al., disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, 1982), rather the virus is horizontally transmitted via suggesting that Mrvi1 is speci®cally targeted by MRV. transplacental infection of implantation stage embryos Mrvi1 encodes a novel protein with homology to Jaw1, a (Bedigian et al., 1993). The BXH2 B-ecotropic virus is lymphoid restricted type II membrane protein that thought to have been generated by a rare recombina- localizes to the endoplasmic reticulum. MRV integration tion event between two defective endogenous ecotropic occurs at the 5' end of the gene between two proviruses, Emv1 and Emv2 inherited from the C3H/ dierentially used promoters. Within hematopoietic cells, HeJ and C57BL/6J parents, respectively (Jenkins et al., Mrvi1 expression is restricted to megakaryocytes and 1982). The susceptibility of BXH2 mice to myeloid some myeloid leukemias. Like Jaw1, which is down- leukemia is genetically determined as infection of other regulated during lymphoid dierentiation, Mrv1 is BXH RI strains with the BXH2 B-ecotropic virus downregulated during monocytic dierentiation of produces either no leukemias or primarily B-cell BXH2 leukemias. Taken together, these data suggest leukemias (Bedigian et al., 1993). that MRV integration at Mrvi1 induces myeloid Several disease genes have been identi®ed by leukemia by altering the expression of a gene important proviral tagging in BXH2 mice. The genes include a for myeloid cell growth and/or dierentiation. Experi- tumor suppressor gene (Nf1) (Buchberg et al., 1990; ments are in progress to test whether Mrvi1 is a tumor Largaespada et al., 1995), the Myb proto-oncogene suppressor gene. (Buchberg et al., 1990; our unpublished results), two class I homeobox genes (Hox7 and Hox9) (Nakamura Keywords: myeloid leukemia model; retroviral inser- et al., 1996), and a Pbx1-related homeobox gene, tional mutagenesis; common sites of integration Mesi1 (Moskow et al., 1995; Nakamura et al., 1996a). Two of these genes, Nf1 and Hoxa9 are also involved in human myeloid leukemia (Shannon et al., 1994; Nakamura et al., 1996b; Borrow et al., 1996). As expected, nearly all of the viral integrations at Myb, Hoxa7, Hoxa9,andMeis1 loci are B-ecotropic viruses. This is not the case, however, for the viruses located at Nf1. In this case, approximately half of the viral *Correspondence: JD Shaughnessy Jr insertions represent a novel defective non-ecotropic Received 15 August 1998; accepted 25 August 1998 virus (Cho et al., 1995). Sequence analysis showed that Mrvi1, a common MRV integration site in BXH2 leukemias JD Shaughnessy et al 2070 the defective virus carries two large deletions, a 1.8 kb MRV genome, integration of MRV into the same deletion in pol and a 1.6 kb deletion in env. Similar EcoRI fragment in dierent tumor DNAs would deletions have been identi®ed in another murine produce comigrating MRV-cellular DNA fragments. retrovirus, the murine AIDS (MAIDS) virus (Aziz et Among the 205 BXH2 tumors analysed, 21 (*10%) al., 1989; Chattopadhyay et al., 1989). Because of this contained MRV proviruses (data not shown) and three structural similarity, this new virus has been designated of the 21 MRV-positive tumors, 87-15, 87-260 and 19 MRV (MAIDS-related virus) (Cho et al., 1995). contained comigrating EcoRI fragments (Figure 1, left Unblot analysis (hybridization to dried agarose gels; panel). The EcoRI fragment from tumor 87-15 was Tsao et al., 1983) with oligonucleotide probes that span subsequently cloned and a unique sequence probe, the MRV env and pol deletions showed that most p15.8.1.1. isolated from the cellular ¯ank (Figure 2). inbred mouse strains carry endogenous MRV-related Hybridization of p15.8.1.1. to Southern blots of proviruses (Cho et al., 1995). BXH2 mice carry three EcoRI-digested DNA from tumor 87-260 and 19 endogenous MRV-related proviruses. One provirus, indicated that tumor 19, but not tumor 87-260, had a Mrv5 maps to the Y chromosome and is carried by viral integration in the same EcoRI fragment as tumor most inbred strain males. The other two proviruses, 87-15 (Figure 1, right panel). The EcoRI fragment Mrv1 and Mrv4, are autosomal. Sequence analysis from tumor 19 was also cloned and restriction enzyme indicated that Mrv1 is the source of the virus present at maps of this clone, as well as clone 87-15, showed that Nf1 (Cho et al., 1995). Transmission of the Mrv1- the two MRV proviruses were integrated 3 kb apart encoded virus to myeloid tumor cells likely involves and in the opposite transcriptional orientation (Figure rescue of Mrv1-encoded virus by a B-ecotropic virus 2). This common site of MRV integration has been helper, which is predicted to occur only rarely. This designated Mrvi1 (MRV integration site 1). B-ecotropic may explain why only *12% of BXH2 leukemias virus did not target Mrvi1 in the 205 B-ecotropic virus- harbor MRV proviruses (Cho et al., 1995). positive BXH2 leukemias analysed (data not shown), A number of models could account for the selection suggesting that Mrvi1 is speci®c for MRV. of somatic MRV proviruses at Nf1. One model that is particularly intriguing suggests that this selection stems Mrvi1 maps to chromosome 7 from the fact that Nf1 is a tumor suppressor gene and both alleles of Nf1 need to be inactivated for leukemia The murine chromosomal location of Mrvi1 was to occur. If the ®rst Nf1 allele is inactivated by the determined by interspeci®c backcross analysis using integration of a non-defective B-ecotropic virus then progeny derived from matings of [(C57BL/6J x Mus this would establish viral interference and make it spretus)F1 X C57BL/6J] mice. This interspeci®c back- dicult for a second viral infection of the same cross mapping panel has been typed for over 2700 loci leukemic cell to occur. However, if the ®rst integration that are well distributed among all the autosomes as were an MRV provirus then viral interference would well as the X chromosome (Copeland et al., 1993). not occur. Consistent with this hypothesis, all BXH2 C57BL/6J and M. Spretus DNAs were digested with leukemias characterized to date that have two viral several restriction enzymes and analysed by Southern integrations at Nf1 contain an MRV provirus blot hybridization for informative restriction fragment integrated in one allele and a B-ecotropic virus length polymorphisms (RFLPs) using probe p15.8.1.1. integrated in the second allele (Cho et al., 1995). The presence or absence of one of these polymorph- To determine whether there are other MRV common integration sites in BXH2 tumors that may harbor tumor suppressor genes, we screened more than 200 BXH2 tumors for new common sites of MRV integration. These studies identi®ed one new common MRV integration site, Mrvi1 that was targeted by MRV in two BXH2 leukemias. Subsequent studies showed that Mrvi1 encodes a novel protein with limited homology to the lymphoid restricted protein, Jaw1 and that like Jaw1, whose expression is downregulated during lymphoid differ- entiation, Mrvi1 expression is downregulated during myeloid dierentiation. Mrvi1 represents a potentially new tumor suppressor gene involved in mouse and human myeloid leukemia.