Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis

Harry C. Hwang*†, Carla P. Martins†‡, Yvon Bronkhorst‡, Erin Randel*, Anton Berns‡, Matthew Fero*, and Bruce E. Clurman§¶

*Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109; ‡Division of Molecular Genetics and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121 1066 CX, Amsterdam, The Netherlands; and §Department of Medicine, University of Washington School of Medicine, Seattle, WA 98104

Communicated by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, June 14, 2002 (received for review May 6, 2002) The p27Kip1 is a cyclin-dependent kinase inhibitor that tumor suppressors such as or p19ARF (10, 11). However, blocks cell division in response to antimitogenic cues. p27 expres- p27Ϫ͞Ϫ mice develop tumors at a greater frequency than do sion is reduced in many human cancers, and p27 functions as a p27ϩ͞ϩ animals when challenged with radiation or chemical tumor suppressor that exhibits haploinsufficiency in mice. Despite carcinogens, and p27ϩ͞Ϫ animals have a tumor frequency the well characterized role of p27 as a cyclin-dependent kinase intermediate between p27ϩ͞ϩ and p27Ϫ͞Ϫ animals (12). Fur- inhibitor, its mechanism of tumor suppression is unknown. We thermore, loss of p27 in mice cooperates with deletion of the used Moloney murine leukemia virus to induce lymphomas in Pten tumor suppressor in the development of prostate cancer, .(p27؉͞؉ and p27؊͞؊ mice and observed that lymphomagenesis and this cooperativity also exhibits p27 haploinsufficiency (13 was accelerated in the p27؊͞؊ animals. To identify candidate This association of reduced (but not absent) p27 expression with oncogenes that collaborate with p27 loss, we used a high-through- tumor-prone phenotypes in mice supports the idea that reduced put strategy to sequence 277 viral insertion sites derived from two p27 expression in human cancers may have similar consequences. ؊͞؊ distinct sets of p27 lymphomas and determined their chromo- p27 expression does not uniformly correlate with markers of somal location by comparison with the Celera and public (Ensembl) cell proliferation in tumors, and its mechanism of tumor sup- mouse genome databases. This analysis identified a remarkable pression remains unclear. We thus sought to identify oncogenes number of putative protooncogenes in these lymphomas, which that collaborate with p27 loss to characterize the transformation included loci that were novel as well as those that were overrep- pathways involved with p27-associated tumorigenesis. We used -resented in p27؊͞؊ tumors. We found that Myc activations oc -؊͞؊ ؉͞؉ Moloney murine leukemia virus (M-MuLV) to induce lympho curred more frequently in p27 lymphomas than in p27 mas in p27Ϫ͞Ϫ and p27ϩ͞ϩ mice. M-MuLV is a nonacute tumors. We also characterized insertions within two novel loci: retrovirus that induces neoplasms through insertional mutagen- (i) the 2 (Jundp2), and (ii) an X-linked esis (14). Proviral integration within host cell DNA activates termed Xpcl1. Each of the loci that we found to be frequently cellular protooncogenes by placing them under the control of involved in p27؊͞؊ lymphomas represents a candidate oncogene viral transcriptional regulatory elements. The principle advan- collaborating with p27 loss. This study illustrates the power of tage of this strategy is that the M-MuLV proviruses serve as high-throughput insertion site analysis in cancer gene discovery. molecular tags that allow for identification of activated that are usually close to the proviral insertion. Because viral utations in genes that regulate the are among the insertions occur randomly within host DNA, common sites Mmost common genetic changes in cancer cells (1). Cell of viral insertion in multiple independent tumors indicate cycle genes that promote cell proliferation are often found as strong biologic selection for these specific integrations. Common dominant oncogenes that have acquired gain-of-function muta- insertion sites (CIS) are thus extremely likely to harbor tions in cancer cells (e.g., cyclin D1). In contrast, components of protooncogenes. the cell cycle machinery that normally inhibit cell division act as In a landmark study, Copeland and coworkers combined tumor suppressors and are frequently inactivated in cancer cells ͞ insertional mutagenesis with functional genomics and reported (e.g., p53). The p27 protein is a member of the Cip Kip family a large-scale analysis of cloned proviral insertion sites that of cyclin-dependent kinase inhibitors and mediates cell cycle identified more than 90 candidate cancer genes in leukemias arrest in response to diverse antimitogenic signals (2). Many arising in AKXD and BXHD mice (15). M-MuLV has also been human cancers show reduced or absent p27 expression compared used extensively to characterize cooperation between oncogenes with normal tissues, and low p27 expression correlates with poor in multistep tumorigenesis. For example, M-MuLV infection of patient outcome in a range of primary tumors (3–5). Unlike E␮-Myc transgenic mice led to the characterization of several classic tumor suppressors, intragenic mutations leading to ho- novel oncogenes that cooperate with Myc activation during mozygous p27 inactivation are very rare in human cancers, and lymphomagenesis (16, 17). In these experiments, lymphomagen- the mechanisms underlying p27 loss in these tumors appear to be esis was accelerated in the E␮-Myc mice compared with the primarily posttranscriptional (6). Thus despite a large body of wild-type animals, because the Myc transgene provided the first data correlating p27 abundance with tumorigenesis, the lack of mutations within the p27 gene in tumors has made it difficult to step in a multistep pathway leading to lymphomas. unambiguously establish that p27 functions as a tumor suppres-

sor in humans. Abbreviations: CIS, common insertion sites; M-MuLV, Moloney murine leukemia virus; In mice, however, p27 clearly functions as a tumor suppressor FHCRC, Fred Hutchinson Cancer Research Center; I-PCR, inverse PCR; EST, expressed that exhibits haploinsufficiency. p27 knockout mice exhibit a sequence tag; LTR, long-terminal repeat. syndrome of gigantism, pituitary and lymphoid hyperplasia, and Data deposition: The sequences reported in this paper have been deposited in the GenBank Ϫ͞Ϫ database (accession nos. AF524047–AF524260 and AY126276–AY126338). female sterility (7–9). Although p27 mice develop pituitary GENETICS adenomas with 100% penetrance, they do not exhibit a wide- †H.C.H. and C.P.M. contributed equally to this work. spread cancer syndrome like those found in mice nullizygous for ¶To whom reprint requests should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.162356099 PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11293–11298 Downloaded by guest on September 28, 2021 We found that M-MuLV-induced lymphomagenesis was greatly accelerated in p27-null mice compared with wild-type animals. We analyzed known M-MuLV targets and found that integrations leading to activation of Myc-related genes occurred more often in p27-null animals, thus identifying Myc activation as an event that may cooperate with p27 loss in lymphomagen- esis. To identify additional p27-complementing oncogenes, we performed a large-scale analysis in which 277 insertion sites derived from two independent panels of p27-null lymphomas were sequenced and compared with the mouse genome data- bases. This analysis reveals a remarkable number of CIS within this group of lymphomas and illustrates the power of high- throughput insertional mutagenesis using the mouse genome sequence in cancer gene discovery. Several of the CIS we identified were overrepresented in the p27-null tumors. Two of these sites were analyzed in further detail: (i) Jundp2, and (ii) an X-linked locus termed Xpcl1 (X-linked p27-complementing locus 1. To our knowledge, this is the first report that Jundp2 functions as a protooncogene. Ϫ͞Ϫ Materials and Methods Fig. 1. Acceleration of lymphomagenesis in p27 mice infected with M-MuLV. (A) Survival of p27Ϫ͞Ϫ, p27ϩ͞Ϫ, and p27Ϫp27ϩ͞ϩ mice is plotted .M-MuLV Infection of p27؉͞؉ and p27؊͞؊ Mice. For panel one against weeks after infection with M-MuLV lymphomas, mice ϩ͞Ϫ for the p27 gene were generated as previously described in C57Bl6 and 129 strains (9). Crosses ϩ͞Ϫ between inbred F0 p27 parental mice were performed to expression levels for Jundp2, Fos, and AI464896 EST were ͞ ϫ ͞ generate F1 C57 B6J 129 Sv hybrid littermates that were normalized to Gapdh expression. Probe and primer sequences p27ϩ͞ϩ, p27Ϯ,orp27Ϫ͞Ϫ. Newborn pups were injected i.p. are available on request. with M-MuLV (1 ϫ 107 pfu) [Dusty Miller, Fred Hutchinson Cancer Research Center (FHCRC)] and genotyped as described Immunoperoxidase Staining of Frozen Tumor Sections. Six-micro- (9). Panel two lymphomas were generated in a similar fashion as meter sections of frozen tumor samples were prepared, and then described (18). Animals were followed for the development of immunocytochemistry was performed by using antibodies to tumors and euthanized when they developed signs of morbidity. CD3, CD4, CD8, and CD45R͞B220 (PharMingen clones L3T4, At necropsy, tumor tissues were snap frozen in liquid nitrogen. Ly-2, 17A2, and RA3–6B2, respectively). Slides were fixed in 10% normal buffered formalin 10 min and then incubated in the DNA Isolation and Southern Analysis. Genomic DNA was isolated following solutions: PBS, 5 min; 100% methanol, 5 min; acetone, from frozen M-MuLV-generated tumors and Southern blots 3 min; and PBS ϫ3 for 10 min. Slides were blocked in 2% normal prepared (17). Southern blots were then hybridized to [␣-32P]- goat serum for 10 min followed by incubation in 1° Ab for 1 hr labeled probes generated by random priming. Final wash steps and then 2° Ab for 30 min (all incubations were performed at were performed in 0.1ϫ SSC and 0.1% SDS at 60°C. Probe and 25°C). Signals were then visualized by using the ABC elite kit primer sequences (where applicable) are available on request. (Vectastain, Vector Laboratories).

Insertion Site Cloning and Sequencing. Inverse PCR (I-PCR): Xpcl1 Genomic Cloning. A mouse genomic lambdaDash II (Strat- I-PCR was performed as previously described (15, 17). I-PCR agene) DNA was obtained from P. Soriano (FHCRC). The primers: SacI, KpnI, and HhaI I-PCR; AB827, AB946, and phage were grown in ER1647, and nylon replica filters were AB949; 5Ј SacII I-PCR, SacII5 (5Ј-CCGTTAAACAGGGAAC- generated and hybridized to an Xpcl1 probe. Positive plaques TAGGGT), AB827, AB949; 3Ј SacII I-PCR, SacII3 (5Ј-CTC- were isolated, purified, and subcloned into pBluescript and CCAATGAGTGCCGGGCCGA), AB946, and AB947 (5Ј-AG- sequenced by using T7 and SP6 primers. TGATTGACTACCCGTCAGCG-3Ј) (15, 17). PCR products were subcloned by using Topo cloning (Invitrogen). All sequenc- Results ing was performed in the FHCRC automated sequencing shared M-MuLV-Induced Lymphomagenesis Is Accelerated in p27؊͞؊ resource. Animals. We infected neonatal p27Ϫ͞Ϫ, p27Ϯ, and p27ϩ͞ϩ pups with M-MuLV. Mice were killed when moribund, and Splinkerette Method. For panel two proviral flanking sequences, tissue was banked for molecular and biochemical studies. Lym- tumor DNA was digested with Sau3AI, ligated to a Splinkerette phomagenesis was significantly accelerated in p27Ϫ͞Ϫ animals adapter, and amplified by three PCR steps by using long- (median survival 18 weeks) compared with p27ϩ͞ϩ mice (me- terminal repeat (LTR) and Splinkerette-specific primers, which dian survival 35 weeks) (P Ͻ 0.001; Fig. 1). The p27ϩ͞Ϫ mice are available on request (19). The resulting PCR fragments were had a survival curve that was moderately accelerated compared sequenced and BLASTed against mouse genome databases. with p27ϩ͞ϩ mice, but this did not reach statistical significance (P Ͼ 0.1). Immunophenotypic and histologic analyses demon- Real-Time PCR. Quantitative real-time PCR was performed by strated that lymphomas were almost exclusively of T cell origin. using an ABI PRISM 7700 sequence detector (Applied Biosys- The lymphomas included CD4ϩCD8ϩ, CD4ϩCD8Ϫ, tems). Triplicate 50-␮l reactions containing 5 ␮l of tumor or CD4ϪCD8ϩ, and CD4ϪCD8Ϫ immunophenotypes. There control thymic cDNA, 25 ␮lofϫ2 Taqman Universal Master were no significant differences in the frequency of lymphoma mix, probe (0.1 ␮M final concentration), and primers (0.2 ␮M immunophenotypes between the p27Ϫ͞Ϫ and p27ϩ͞ϩ groups final concentration) for Jundp2, Fos, AI464896 expressed se- (data not shown). quence tag (EST), and mouse Gapdh were prepared. Samples were cycled in 96-well plates per manufacturer’s instructions and Increased Frequency of Myc Activations in p27؊͞؊ Lymphomas. We data analyzed by using SDS Software (Applied Biosystems). The screened 25 p27Ϫ͞Ϫ and 25 p27ϩ͞ϩ lymphoma DNAs by

11294 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.162356099 Hwang et al. Downloaded by guest on September 28, 2021 Table 1. Involvement of M-MuLV insertion sites in p27-null and tion of the viral insertions in these lymphomas may have p27 wild-type lymphomas by Southern analysis contributed to their neoplastic progression. Locus p27-null tumors p27 wild-type tumors P value We identified three classes of CIS. The first class represented insertions near or within known M-MuLV protooncogene tar- Myc* 14͞25 (56%) 4͞25 (16%) 0.0031 gets and includes Notch1, Rasgrp1, Gfi1͞Evi5, Myb, NMyc1, Pim1 7͞25 (28%) 6͞25 (24%) – Myc͞Pvt1, and Pim1 loci (sites 1–7; Table 2) (17, 20–24). This Gfi1 1͞25 (4%) 2͞25 (8%) – group of insertions contained the most commonly identified sites Jundp2 7͞25 (28%) 2͞25 (8%) 0.058 and comprised over half of all sequence tags found within CIS Xpcl1 7͞25 (28%) 1͞25 (4%) 0.0224 (55͞96, 58%). ͞ *Includes Myc, Nmyc1, and Pvt1 loci. The next most frequent group of CIS (24 96, 25%) overlapped with either known or predicted genes that have not been previously identified as CIS (sites 8–15; Table 2). Examples Southern blotting for involvement of previously defined M- include Jundp2 and a novel SH3 domain-containing protein. The MuLV CIS. A M-MuLV U3 LTR probe demonstrated that the final class of insertions fell between annotated genes (20–200 kb distance). These insertions comprised Ϸ18% of all CIS identified lymphomas contained between 2 and 12 clonal viral insertions, ͞ and the number of insertions did not vary significantly between (17 96, sites 16–22; Table 2). the p27Ϫ͞Ϫ and p27ϩ͞ϩ groups (data not shown). We then For CIS that were near or within genes (classes 1 and 2), it is determined the frequency of insertions within several known likely that the indicated genes are the targets of the integrations, although not all of the predicted genes have been experimentally CIS in these lymphomas. Pim1 and Gfi1 insertions occurred with determined to represent bona fide genes. For CIS that fell similar frequencies in p27Ϫ͞Ϫ and p27ϩ͞ϩ mice (Table 1). between genes (class 3), the target is less clear, particularly Neither the total number of viral insertions per tumor nor the because proviruses in this class are quite distant from the closest frequency of CIS such as Gfi1 and Pim1 was increased in the Ϫ͞Ϫ mapped genes. In all cases, however, the putative targets must be p27 lymphomas, indicating that p27 loss does not accelerate validated by direct examination of mRNA in tumors containing lymphomagenesis by increasing the total number of viral inser- the relevant viral insertions. tions in target cells. In contrast, insertional activation of Myc Ϫ͞Ϫ ϩ͞ϩ occurred more frequently in p27 compared with p27 Identification of Jundp2 as a Protooncogene That Is Frequently ϭ mice (Table 1; P 0.0031). This overrepresentation of Myc Activated in p27؊͞؊ Lymphomas. Site 14 (Table 2) represented Ϫ͞Ϫ activation in p27 lymphomas strongly suggests that Myc insertions within and flanking Jundp2. Jundp2 is a member of the cooperates with p27 loss. In support of this idea, synergism AP-1 family of b-zip transcription factors and was first cloned between Myc activation and p27 loss was also observed in through its physical interaction with the Jun protein (24). More independent models of Myc-associated lymphomagenesis by recently, Jundp2 overexpression was found to attenuate p53- Martins and Berns, who observed acceleration of T and B cell dependent in response to UV irradiation (25). Because lymphomas when p27Ϫ͞Ϫ animals were crossed with strains Jundp2’s known biologic activities suggested a link to tumori- containing H2K-Myc and E␮-Myc transgenes, respectively (19). genesis, site 14 was chosen for further analysis. By using a Jundp2-specific probe, we detected proviral rearrangements in a Identification of Putative p27-Complementing Oncogenes by High- total of seven p27Ϫ͞Ϫ and two p27ϩ͞ϩ lymphomas (Table 1). Throughput Insertion Site Analyses. To identify novel oncogenic We have mapped the location of these insertions within Jundp2 loci in the p27Ϫ͞Ϫ tumors, we applied large-scale sequencing of by using the Celera database in combination with Southern genomic DNA adjacent to proviruses. The lymphomas used for blotting and grouped them into three clusters (A, B, and C; Fig. this analysis were obtained from two independent panels of 2). As shown in Fig. 2, the proviral insertions in this locus p27Ϫ͞Ϫ lymphomas. Panel one consisted of the 25 p27Ϫ͞Ϫ included integrations within (clusters A and B) and flanking lymphomas described above, whereas panel two was composed (cluster C) Jundp2. of 45 p27Ϫ͞Ϫ M-MuLV-induced lymphomas described by Mar- We next examined each cluster for evidence of Jundp2 acti- tins and Berns (18). The two panels differed somewhat with vation. For clusters A and B, quantitative real-time PCR analysis regard to strain background, tumor latency, and surface markers. using a Jundp2 1 probe revealed that five of seven panel one More importantly, however, the panels were quite similar in that lymphomas with Jundp2 insertions contained increased amounts in both lymphoma cohorts, p27 loss resulted in acceleration of of Jundp2 mRNA compared with lymphomas without Jundp2 M-MuLV-induced lymphomagenesis, as well as a high frequency insertions. In contrast, these same tumors did not exhibit in- of Myc activations. The insertion site sequences were obtained by creased expression of Fos, the closest linked gene to Jundp2 (Fig. 2C). Thus the insertions within the Jundp2 locus did not lead to I-PCR for panel one lymphomas and by an alternative PCR generalized increased expression of all nearby genes but specif- strategy (Splinkerette; see Materials and Methods) for panel two ically enhanced Jundp2 expression. Two p27-null tumors with lymphomas. After the sequence tags were obtained, they were Jundp2 insertions did not show increased Jundp2 mRNA levels mapped by using both the Celera and public (Ensembl) mouse (tumors 968 and 168). In the case of tumor 168, the viral insertion genome databases. occurred downstream of the exon 1 probe sequence used for We sequenced a total of 277 insertion sites from these 70 Ϫ͞Ϫ measuring mRNA expression, and the truncated transcript does p27 lymphomas. A complete list of these insertion sites is not contain the first exon (see below). Southern analysis revealed provided in Table 3, which is published as supporting informa- that the rearranged Jundp2 band for 968 was subclonal, thus the tion on the PNAS web site, www.pnas.org. We used the chro- low levels of Jundp2 mRNA in this tumor may result from this mosomal location of these sites to identify candidate CIS (clones subclone being underrepresented in this tumor sample. Ϸ that fell within 100 kb of one another were considered to We next characterized Jundp2 transcripts in two tumors represent a CIS). The 21 CIS revealed by this analysis are (tumors 168 and 83) with integrations within cluster B to presented in Table 2. The combination of the two tumor panels determine whether these insertions also caused Jundp2 activa- led to an increase in the number of CIS identified, as well as an tion. The location and orientation of these proviruses suggested

increased frequency of insertions within sites identified in both that they might activate Jundp2 through promoter insertion, in GENETICS panels. Remarkably, 35% (96͞277) of all insertion sites repre- which transcription initiates within the viral LTR and then sented putative CIS, indicating that a surprisingly large propor- splices to Jundp2 . Indeed, by using a directed PCR

Hwang et al. PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11295 Downloaded by guest on September 28, 2021 Table 2. CISs identified in p27-null lymphomas Celera annotation† Ensembl annotation‡

Site No. Location, mCG Location, ENSMUSG no. tumors Chr* Mb no. Gene description Mb no. Gene description

Clusters involving known 1 2 2 22.663–22.664 18747 Notch1 26.754–26.755 26923 Notch1 proviral integration sites 2 3 2 115.320–115.401 14557 Rasgrp1 118.271–118.353 27347 Rasgrp1 3 20 5 100.520–100.577 19138 Gfi1͞Evi5 105.372–105.428 29275 Gfi1͞Evi5 4 5 10 17.868–17.987 2825 Myb 21.003–21.130 19982 Myb 5 3 12 9.894–9.895 19753 NMyc1 13.030–13.031 37169 Nmyc1 6 20 15 56.913–57.145 1625 Myc͞Pvt1 62.430–62.663 22346 Myc͞Pvt1 7 2 17 22.055–22.060 21141 Pim1 28.704–28.708 24014 Pim1 Clusters involving known 8 5 2 26.789–26.854 19005 Unknown 30.971–31.038 26859 Unknown or putative genes 9 2 2 161.340–161.374 5444 Pkig 164.575–164.608 35268 Pkig 5448 TDE1 homologue 17707 TDE1 homologue 10 2 3 86.406–86.409 5011 Rorc 94.722–94.723 28150 Rorc 11 2 4 130.357–130.361 20817 veg homologue 131.234–131.238 37543 veg homologue 12 2 7 49.367–49.409 13842 Igf1r 57.651–57.693 05533 Igf1r 13840 Pyr.-Carboxylate peptidase 30553 Pyr.-carboxylate peptidase 13 4 10 17.749–17.832 2824 SH-3 Domain protein 20.885–20.968 37571 SH-3 domain protein 14 5 12 75.414–75.511 5743 Jundp2 80.242–80.339 34271 Jundp2 15 2 19 32.634–32.650 49335 Unknown 38.507–38.522 24999 Unknown Clusters between known 16 2 3 46.819–46.820 1650 KIAA1816 homologue 52.521–52.522 37078 KIAA1816 homologue or putative genes§ 1648 Foxo1 37068 Foxo1 17 2 6 125.033–125.034 13072 Tpi 128.163–128.164 37997 Unknown 13058 Calm2 30349 40S ribosomal protein 18 5 7 94.633–94.642 51745 Rras2 103.790–103.797 38142 Rras2 2291 Copb1 30754 Copb1 19 2 9 67.173–67.175 16396 Tcf12 72.772–72.775 32228 Tcf12 130222 Ub. carboxy extension protein 38657 Ribosomal protein L40e 20 2 11 76.252–76.253 1336 Diphthamide DPH2 protein 75.809–75.811 38268 Diphthamide DPH2 protein 1330 Replication protein A subunit 00751 Replication protein A subunit 21 4 X 27.438–27.440 14311 Pou transcription factor 38.350–38.354 36050 Repeat family 3 gene 50918 Gpc3 31120 Gpc3

Pyr., pyrrolidone; Ub., ubiquitin. *. †Celera annotation based on the Celera Mouse Genome Database (CMGD Release 12). ‡Ensembl annotation based on the Public Mouse Genome Assembly (Ver. 6.3a.1). Both the Celera and Ensembl annotations include megabase (Mb) location, gene number, and annotated gene description. §For clusters falling between genes (sites 16–21), the nearest annotated genes on either side of the cluster are shown.

strategy, we cloned chimeric transcripts from cDNA derived from tumors 168 and 83 (data not shown). In each case, the transcript initiated in the LTR and spliced into Jundp2 exon 3 and 4 sequences. Finally, we examined Jundp2 expression by Northern blot in the three panel two lymphomas that contained cluster C insertions. The viruses in these lymphomas are all oriented in the opposite transcriptional orientation from Jundp2, suggesting a mechanism of activation by enhancement of the Jundp2 promoter. Each of these lymphomas showed overexpres- sion of Jundp2 mRNA (data not shown). Thus, our studies revealed that insertions within each of the three clusters (A, B, and C) led to overexpression of Jundp2 mRNA.

Site 21 Represents a Tightly Clustered X-Linked CIS That Is Enriched in -p27؊͞؊ Lymphomas. Site 21 was isolated from 4 p27Ϫ͞Ϫ lym phomas (three panel one lymphomas and one panel two lym- phoma) (Table 2). All of these insertions fell within 3 kb of one another. Because such tight clustering is usually indicative of a Fig. 2. Jundp2 is a target for retroviral insertion in M-MuLV-induced nearby protooncogene, we analyzed site 21 in more detail. lymphomas. (A) Genomic structure of the Jundp2 locus. The position and By using a site 21-specific probe, we detected rearrangements orientation of sequenced viral insertions (arrows) are shown (see text). Boxes in 28% (7͞25) of the panel one p27Ϫ͞Ϫ lymphomas versus 4% denote the four Jundp2 exons (coding regions are hatched), and EcoRV sites ͞ ϩ͞ϩ are indicated (R5). (B) Expression of Jundp2 mRNA in M-MuLV-induced tumors (1 25) of the panel one p27 lymphomas (Table 1). The and normal thymus (Thy). Quantitative real-time PCR was performed by using difference in frequency of involvement between the p27Ϫ͞Ϫ a first exon Jundp2 probe. (C) Expression of Fos mRNA in M-MuLV-induced and p27ϩ͞ϩ lymphomas was statistically significant (P ϭ 0.022), tumors as determined by quantitative real-time PCR. suggesting that this locus cooperates with p27 loss.

11296 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.162356099 Hwang et al. Downloaded by guest on September 28, 2021 mice against the Celera and public mouse genome databases, we were able to identify a larger number of CIS than were found in each panel individually. Several of these common sites appear to be novel and enriched in p27Ϫ͞Ϫ versus p27ϩ͞ϩ lymphomas. These sites likely harbor genes that on gain or loss of function collaborate with p27 loss. Multiple studies have suggested that the ability of Myc to abrogate p27 function is a critical mechanism involved with Myc-induced transformation (26–29). We were thus surprised to find not only that Myc activations occur in p27Ϫ͞Ϫ lymphomas, but they were found more frequently in the p27Ϫ͞Ϫ group. This strong selection for Myc insertional activation in lymphomas lacking p27 indicates that p27 ‘‘override’’ is not the primary mechanism of Myc function in lymphomagenesis. In agreement with this result, Martins and Berns have studied in detail the synergy between Myc activation and p27 loss and found strong cooperation between these two events in T and B cell transgenic Myc models in addition to M-MuLV-induced lymphomas (18). Thus the cooperation between Myc and p27 loss is not unique to M-MuLV-induced lymphomagenesis, but rather is a common feature of p27-associated lymphomas and perhaps other cancers. We have identified Jundp2 as a target for insertional activation in lymphomas. Although insertions within Jundp2 are enriched in p27Ϫ͞Ϫ lymphomas, Jundp2 activations are not restricted to Fig. 3. Xpcl1 is a CIS in p27Ϫ͞Ϫ lymphomas and linked to a novel thymic EST. this group of lymphomas. Several lines of evidence support the (A) Genomic map of the Xpcl1 locus and closest Celera annotated genes. The identification of Jundp2 as the target gene of the insertions relative position of the Xpcl1 genomic clone and the sequences identical to the within this region of chromosome 12: (i) most insertions fell mouse AI464896 EST are shown (EST; see text). (B) Overexpression of mRNA directly within Jundp2;(ii) Jundp2-associated insertions were homologous to the AI464896 EST as determined by quantitative real-time PCR. associated with overexpressed Jundp2 mRNA; and (iii) expres- S, spleen; T, thymus. sion of the nearby Fos gene was unaffected. Many of the integrations caused truncation of the Jundp2 protein, although in those cases the b-zip region of the protein was left intact. It We used the Celera database to assemble a genomic map of is possible that these tumor-specific truncated Jundp2 alleles are the site 21 insertion region (Fig. 3A). This region falls within the selected because they are more potent transforming . mouse X chromosome and is highly syntenic with human Xq26. However, the fact that many of the lymphomas contained No known oncogenes or tumor suppressor genes have been insertions that leave the Jundp2 coding sequence intact indicates mapped in this region. The positions of the four insertions that that truncation is not required for the oncogenic activity of were sequenced are indicated. These insertions fell in a region Jundp2. We found one tumor (772, Fig. 2B) with elevated Jundp2 relatively underrepresented by known genes. However, there are expression but without a Jundp2-associated provirus. In this case, gaps remaining in the Celera sequence. The nearest known gene it is likely that either a Jundp2-associated provirus was outside Ϸ Ј is Gpc3, which falls 200 kb 3 to the insertion cluster. There is of the region encompassed by the Southern analysis presented in Ϸ also a hypothetical POU domain transcription factor gene 200 Fig. 2 (e.g., in cluster C) or it is also possible that Jundp2 Ј kb 5 of the insertion cluster. However, there does not appear to expression was elevated in this lymphoma by a mechanism other be a human X-chromosomal homologue of this predicted gene, then insertional activation. and its functional status is unknown. Karin and coworkers have reported that Jundp2 expression is Because this region of the X chromosome is incompletely induced by UV-C irradiation, and that Jundp2 represses p53 Ϸ sequenced, we obtained an 20-kb DNA fragment covering this transcription by binding to an atypical AP-1 site in the p53 locus from a mouse genomic library. A region of this clone was promoter (25). It is thus tempting to speculate that insertionally found to contain a sequence identical to a 505-bp expressed activated Jundp2 promotes lymphomagenesis by disabling p53. sequence tag (National Center for Biotechnology Information However, we have no data that directly support this idea. We accession no. AI464896) derived from a mouse thymus library examined p53 expression in panel one p27Ϫ͞Ϫ lymphomas and (Fig. 3A). Quantitative PCR showed that all panel one lympho- were unable to detect p53 protein in those tumors, so we could mas with this insertion site contained elevated levels of the not determine whether p53 abundance was affected by Jundp2 AI464896 transcript, demonstrating that the viral insertions overexpression (data not shown). We also overexpressed Jundp2 activated AI464896 expression (Fig. 3B). However, AI464896 in primary p27ϩ͞ϩ or p27Ϫ͞Ϫ mouse embryo fibroblasts and does not contain an ORF. Three possible explanations are: did not observe phenotypes suggestive of p53 loss, including the (i) this insertionally activated EST may function as a noncoding ability to form colonies and insensitivity to Myc-induced RNA; (ii) this transcript may not be the true target of the apoptosis (H.C.H. and B.E.C., unpublished results). Thus the insertion cluster; or (iii) it may represent only a partial cDNA. mechanism through which Jundp2 promotes tumorigenesis We have designated this locus Xpcl1 (X-linked p27-complement- remains unknown. ing locus 1) until the insertional target within this region is Xpcl1 is an X-linked locus that we found to be enriched in unambiguously identified. p27Ϫ͞Ϫ lymphomas. Integrations within this locus are tightly clustered, strongly suggesting a common target gene. We have Discussion identified a candidate transcript corresponding to a mouse EST We used proviral tagging in p27Ϫ͞Ϫ mice to identify protoon- sequence that is encoded near the insertion sites and overex-

cogenes that cooperate with p27 loss during multistep lym- pressed in all lymphomas containing Xpcl1 insertions. There is GENETICS phomagenesis. By screening insertion site sequences from two significant sequence conservation between the murine Xpcl1 distinct panels of M-MuLV-induced lymphomas in p27Ϫ͞Ϫ locus and the syntenic human Xq26 region, and sequences

Hwang et al. PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11297 Downloaded by guest on September 28, 2021 homologous to AI464896 and the cloned insertion sites are observed in related screens in different genetic backgrounds. present in the human Xq26 region (data not shown). However, This series includes approximately 2,000 cloned insertion sites expressed human transcripts homologous to AI464896 have not from M-MuLV-induced lymphomas from BXH2, AKXD, E␮- been reported in sequence databases. Because Xpcl1 is located Myc; Pim1Ϫ͞Ϫ; Pim-2Ϫ͞Ϫ, and Cdkn2aϪ͞Ϫ mouse strains (18, within the X chromosome, it is also possible that these insertions 30, 31). The sites that we identified corresponding to previously could inactivate a in a single step. known insertion sites (Table 2, sites 1–7) were also found However, there are no data currently to support the idea that a frequently in these other screens (e.g., Myc, Gfi1, and Rasgrp1). tumor suppressor resides within the Xpcl1 locus. Several of the novel loci that we have described in Table 2 (sites The availability of the completed mouse genome has greatly 8–21) were also cloned frequently in these screens, indicating facilitated the process of cancer gene discovery by insertional that they are not likely to be specifically enriched in the p27Ϫ͞Ϫ mutagenesis. The power of this technique is demonstrated by the background (e.g., site 13, SH3 domain protein). In contrast, large number of previously unrecognized CIS found within the other CIS that we have identified were found only rarely or not p27Ϫ͞Ϫ lymphomas. We arbitrarily defined a region as a at all in these other genetic backgrounds, further supporting the putative CIS if two or more independent insertions occurred idea that they are specifically selected for in the p27Ϫ͞Ϫ within a Ϸ100-kb span of DNA. This restriction may omit true background. Clones corresponding to Jundp2 and Xpcl1 loci CIS that activate a common target gene over greater distances were each found only once among the 2,000 sites. Similarly, or falsely include clustered insertions that occurred randomly. several other sites listed in Table 2 were found only once or not This analysis also revealed that a surprisingly high percentage of at all in the larger insertion site collection (sites 8, 10–12, 15, 16, integrations fell within CIS that included both well-characterized 19, and 20; Table 2). Each of these loci thus represents a potential and novel loci. Two distinct mouse genome databases are now oncogene that collaborates with p27 loss in lymphomagenesis. available: the Celera and the public databases. As shown in Table However, as the activated genes within these loci are identified, 2, the data that we have obtained from these two resources have their activity in the setting of p27 loss must be individually tested. been quite similar thus far. However, as these databases improve Finally, although we have focused on CIS that are enriched in in both coverage and annotation, it is likely that each may have p27-null lymphomas as being the most likely genes that coop- unique advantages for analyzing large sets of insertion site data. erate with p27 loss, it is important to realize that CIS found in Although the frequency with which sites were sequenced other backgrounds, as well as p27-null lymphomas, may also largely reflected the frequency of involvement detected by cooperate with p27 loss. Perhaps in these other genetic back- Southern blotting, this was not always the case. For example, the grounds, it is the p27 pathway that has been mutated. Thus by Pim1 locus was a common target in p27Ϫ͞Ϫ tumors (Table 1) comparing the CIS involved in diverse genetic backgrounds, a but was found only twice among the 277 sequence tags (Table 3). defined set of common pathways may be revealed that are The likelihood of identifying specific insertion sites by the PCR involved in the genesis of a large number of cancers. methods we used in this study may thus be influenced by other factors such as the presence of repetitive sequences, restriction We thank Dusty Miller (FHCRC) for providing M-MuLV reagents and site location, etc. Therefore, some CIS may be underrepresented expertise and James Roberts (FHCRC) for advice and support. We also in the collection of insertion site sequences. Using multiple thank Ami Aronheim for providing Jundp2 antibody. B.E.C. is a W. M. Keck Distinguished Young Scholar in Medical Research. This work was techniques to obtain insertion sites, such as the two methods we supported by National Institutes of Health Grant R01 CA84069 (B.E.C.) describe, may increase the total number of CIS identified by and Department of Defense Grant DAMD17-98-1-8308 (B.E.C.), Na- overcoming technical barriers associated with any single method. tional Institutes of Health Training Grant CA80416-05 (H.C.H.), and the We have compared the frequency of site involvement that we Praxis XXI and Gulbenkian Foundations (C.P.M.). H.C.H is an E. observed in p27Ϫ͞Ϫ mice with the utilization of insertion sites Donnall Thomas Scholar of the Jose Carreras Foundation.

1. Sherr, C. J. (1996) Science 274, 1672–1677. 17. Scheijen, B., Jonkers, J., Acton, D. & Berns, A. (1997) J. Virol. 71, 9–16. 2. Sherr, C. J. & Roberts, J. M. (1999) Genes Dev. 13, 1501–1512. 18. Martins, C. P. & Berns, A. (2002) EMBO J. 21, in press. 3. Clurman, B. E. & Porter, P. (1998) Proc. Natl. Acad. Sci. USA 95, 15158–15160. 19. Mikkers, H., Allen, J., Knipscheer, P., Romeyn, L., Vink, E. & Berns, A. (2002) 4. Lloyd, R. V., Erickson, L. A., Jin, L., Kulig, E., Qian, X., Cheville, J. C. & Nat. Genet., in press. Scheithauer, B. W. (1999) Am. J. Pathol. 154, 313–323. 20. Yanagawa, S., Lee, J. S., Kakimi, K., Matsuda, Y., Honjo, T. & Ishimoto, A. 5. Slingerland, J. & Pagano, M. (2000) J. Cell Physiol. 183, 10–17. (2000) J. Virol. 74, 9786–9791. 6. Ponce-Castaneda, M. V., Lee, M. H., Latres, E., Polyak, K., Lacombe, L., 21. Shen-Ong, G. L., Potter, M., Mushinski, J. F., Lavu, S. & Reddy, E. P. (1984) Montgomery, K., Mathew, S., Krauter, K., Sheinfeld, J., Massague, J., et al. Science 226, 1077–1080. (1995) Cancer Res. 55, 1211–1214. 22. Corcoran, L. M., Adams, J. M., Dunn, A. R. & Cory, S. (1984) Cell 37, 7. Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C., 113–122. Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A. & Koff, 23. van Lohuizen, M., Breuer, M. & Berns, A. (1989) EMBO J. 8, 133–136. A. (1996) Cell 85, 721–732. 24. Aronheim, A., Zandi, E., Hennemann, H., Elledge, S. J. & Karin, M. (1997) 8. Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Mol. Cell Biol. 17, 3094–3102. Horii, I. & Loh, D. Y. (1996) Cell 85, 707–720. 25. Piu, F., Aronheim, A., Katz, S. & Karin, M. (2001) Mol. Cell Biol. 21, 3012–3024. 9. Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, 26. Bouchard, C., Thieke, K., Maier, A., Saffrich, R., Hanley-Hyde, J., Ansorge, K., Tsai, L. H., Broudy, V., Perlmutter, R. M., et al. (1996) Cell 85, 733–744. W., Reed, S., Sicinski, P., Bartek, J. & Eilers, M. (1999) EMBO J. 18, 10. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, 5321–5333. C. A., Jr., Butel, J. S. & Bradley, A. (1992) Nature (London) 356, 215–221. 27. Perez-Roger, I., Kim, S. H., Griffiths, B., Sewing, A. & Land, H. (1999) EMBO 11. Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D. & DePinho, R. A. 18, (1996) Cell 85, 27–37. J. 5310–5320. 12. Fero, M. L., Randel, E., Gurley, K. E., Roberts, J. M. & Kemp, C. J. (1998) 28. O’Hagan, R. C., Ohh, M., David, G., de Alboran, I. M., Alt, F. W., Kaelin, Nature (London) 396, 177–180. W. G., Jr., & DePinho, R. A. (2000) Genes Dev. 14, 2185–2191. 13. Di Cristofano, A., De Acetis, M., Koff, A., Cordon-Cardo, C. & Pandolfi, P. P. 29. Yang, W., Shen, J., Wu, M., Arsura, M., FitzGerald, M., Suldan, Z., Kim, D. W., (2001) Nat. Genet. 27, 222–224. Hofmann, C. S., Pianetti, S., Romieu-Mourez, R., et al. (2001) Oncogene 20, 14. Jonkers, J. & Berns, A. (1996) Biochim. Biophys. Acta 1287, 29–57. 1688–1702. 15. Li, J., Shen, H., Himmel, K. L., Dupuy, A. J., Largaespada, D. A., Nakamura, 30. Suzuki, T., Shen, H., Akagi, K., Morse, H. C., Jenkins, N. A. & Copeland, N. G. T., Shaughnessy, J. D., Jr., Jenkins, N. A. & Copeland, N. G. (1999) Nat. Genet. (2002) Nat. Genet., in press. 23, 348–353. 31. Lund, A. H., Turner, G., Trubetskoy, A., Verhoeven, E., Wientjens, E., 16. van Lohuizen, M., Verbeek, S., Scheijen, B., Wientjens, E., van der Gulden, H. Hulsman, D., Russell, R., DePinho, R. A., Lenz, J. & van Lohuizen, M. (2002) & Berns, A. (1991) Cell 65, 737–752. Nat. Genet., in press.

11298 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.162356099 Hwang et al. Downloaded by guest on September 28, 2021