Published OnlineFirst January 12, 2010; DOI: 10.1158/0008-5472.CAN-09-2736 Published Online First on January 12, 2010 as 10.1158/0008-5472.CAN-09-2736 Molecular and Cellular Pathobiology Cancer Research Insertional Mutagenesis in Mice Deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 Reveals Cancer Gene Interactions and Correlations with Tumor Phenotypes Jaap Kool1, Anthony G. Uren1, Carla P. Martins1, Daoud Sie2, Jeroen de Ridder3,4, Geoffrey Turner5, Miranda van Uitert3, Konstantin Matentzoglu1, Wendy Lagcher1, Paul Krimpenfort1, Jules Gadiot1, Colin Pritchard1, Jack Lenz5, Anders H. Lund1, Jos Jonkers3, Jane Rogers6, David J. Adams6, Lodewyk Wessels3,4, Anton Berns1, and Maarten van Lohuizen1 Abstract The cyclin dependent kinase (CDK) inhibitors p15, p16, p21, and p27 are frequently deleted, silenced, or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development, showing that these genes function as tumor suppressors. Here, we describe high-throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also found associations between these loci with gender, age of tumor onset, and lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with single nucleotide polymorphisms associated with chronic lymphocytic leukemia revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association studies. Cancer Res; 70(2); 520–31. ©2010 AACR. Introduction making it difficult to determine which events are causal “driver” mutations and which are incidental “passenger” Recent mutation profiling of human tumors has implic- mutations (reviewed in ref. 1). Retroviral insertional muta- ated hundreds of genes in the pathogenesis of cancer; how- genesis screens performed in mouse models of cancer are ever, the vast majority of these genes are only rarely mutated, useful complements to studies of human tumors because they provide an independent validation of oncogenic muta- Authors' Affiliations: 1Division of Molecular Genetics, The Centre of tions in another organism (2). In these screens, slow-trans- Biomedical Genetics, Academic Medical Center and Cancer Genomics Centre, 2Central Microarray Facility, and 3Division of Molecular Biology, forming retroviruses are used to induce mutations within Netherlands Cancer Institute, Amsterdam, the Netherlands; 4Faculty of somatic tissues (3). Over the lifetime of the mouse, these Electrical Engineering, Mathematics, and Computer Science, Delft mutations accumulate, eventually promoting clonal expan- University of Technology, Delft, the Netherlands; 5Albert Einstein College of Medicine, Bronx, New York; and 6Wellcome Trust Sanger sion of cells bearing multiple oncogenic mutations. Because Institute, Hinxton, United Kingdom viral insertion sites can be easily identified by PCR, a high pro- Note: Supplementary data for this article are available at Cancer portion of the mutations in each tumor can be identified with Research Online (http://cancerres.aacrjournals.org/). relatively little effort. This coverage allows for the identifica- J. Kool and A.G. Uren contributed equally to this work. tion of genetic interactions between mutations at rates that Current address for C.P. Martins: Cancer Research UK, Cambridge are not yet possible from the study of human tumors. Further- Research Institute, Cambridge, United Kingdom. more, the use of mice allows the identification of cancer genes Current address for G. Turner: Department of Pathology, NorthShore that collaborate with specific cancer-predisposing lesions that University HealthSystem, Evanston, Illinois. have been introduced into the germline. Current address for K. Matentzoglu: Trenzyme GmbH, Konstanz, Germany. Cyclin/cyclin-dependent kinase (CDK) complexes pro- Current address for A.H. Lund: Biotech Research and Innovation Centre and mote the progression of cells through the cell cycle. Dereg- Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark. ulation of CDKs is implicated in the progression of a variety Current address for J. Rogers: The Genome Analysis Centre, John Innes Centre, Norwich, United Kingdom. of cancers, including hematologic malignancies (reviewed in ref. 4). Based on structural and functional characteristics, Corresponding Author: Anton Berns or Maarten van Lohuizen, Nether- lands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, the two families of CDK inhibitors can be distinguished: the Netherlands. Phone: 31-20-512-1990; Fax: 31-20-512-2011; E-mail: Ink4 family (p16Ink4a, p15Ink4b,p18Ink4c,andp19Ink4d)and [email protected] or [email protected]. the Cip/Kip family (p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2; re- doi: 10.1158/0008-5472.CAN-09-2736 viewed in ref. 5). The Ink4 proteins specifically inhibit cyclin ©2010 American Association for Cancer Research. D–cdk4-6 complexes and expression of Ink4 genes results in 520 Cancer Res; 70(2) January 15, 2010 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 12, 2010; DOI: 10.1158/0008-5472.CAN-09-2736 Insertional Mutagenesis in CDK Inhibitor–Deficient Mice Ink4b Ink4a aG1-phase cell cycle arrest. p15 and p16 as well as insertion sites (CIS) targeting known cancer genes as well as p19Arf (which shares part of its coding sequence with novel candidate cancer genes. Many of these CISs are specif- p16Ink4a in a different reading frame) are located in the ically mutated in mice lacking one or more CDK inhibitors. same chromosomal region that is frequently deleted in hu- Furthermore, we find CISs correlating with the cell type of man cancer (6). Germline knockout studies have shown origin and latency of the tumor, as well as co-occurring or that loss of p15 and p16 predisposes mice for tumor devel- mutually exclusive combinations of mutations. We also opment, confirming their role as tumor suppressor genes found a significant correlation between the location of single (7–10). nucleotide polymorphisms (SNP) associated with familial The Cip/Kip proteins have a broader spectrum of sub- chronic lymphocytic leukemia (CLL) and CISs, indicating strates, binding to cyclin D–, cyclin E–, and cyclin A–depen- that insertional mutagenesis data may be of use in identify- dent kinase complexes. The p27 and p21 genes are rarely ing disease-associated genes. subject to inactivating mutations in human cancer, but can be inactivated by other mechanisms (reviewed by ref. 5). Mouse studies have clearly indicated that p27 is a tumor sup- Materials and Methods pressor gene (11, 12). Germline disruption of p21 may have both antioncogenic and pro-oncogenic effects depending on Mice strains and induction of tumors by murine leuke- the genetic context (13–15). mia virus infection. Some tumors were described in prior We performed retroviral insertional mutagenesis in mice studies (16–18). p15Ink4b-deficient mice are described in ref. deficient for one or more Ink4 and/or Cip/Kip family mem- (7). Newborn pups were injected i.p. with the supernatant of bers to identify genes that collaborate with absence of these murine leukemia virus (MuLV)–producing NIH3T3s. Animals CDK inhibitors in cancer. Analyzing tumors from 476 mice, were monitored for the development of tumors; moribund we identified 9,117 insertions, finding hundreds of common mice were sacrificed and tumors were isolated. Figure 1. MuLV insertion density over the genome. A, flow chart of the experimental setup. B, insertions from all tumor panels were combined to calculate the insertion density over the genome using a 30-kb kernel (21). Red lines, the cutoff (P = 0.05) for significant insertion density. Green lines, CISs (P < 0.05). Wt, wild-type. www.aacrjournals.org Cancer Res; 70(2) January 15, 2010 521 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 12, 2010; DOI: 10.1158/0008-5472.CAN-09-2736 Kool et al. Figure 2. Selection for mutation of prominent cancer genes in different genotypes. Using Fisher's exact test, we examined whether each CIS is significantly more mutated in each genotype versus every other genotype. P values for each test are represented as a heat map (significance is indicated by the color bar). Green squares, a gene is mutated more frequently in the genotype indicated on the vertical axis versus the genotype on the horizontal axis. Red squares, a gene is more mutated in the genotype on the horizontal axis versus the genotype on the vertical axis. CIS rank and CIS name are indicated above each heat map. A, heat maps for Myc/Pvt1 and Mycn. B, heat maps for Ccnd3 and Ccnd1. C, heat maps for mmu-mir-106-363 and mmu-mir-17-92. Cloning of insertion sites. Ligation-mediated splinkerette Informatics. Sequences were mapped onto National PCRs were performed as described in ref. (19). Two ligations Center for Biotechnology Information mouse build 37 were performed for each tumor, using DNA digested with using exonerate (20). Sequences