Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis

November Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis Irina Vyazunova 0 5 6 Vilena I. Maklakova 0 5 6 Samuel Berman 1 5 6 Ishani De 0 5 6 Megan D. Steffen 0 5 6 Won Hong 0 5 6 Hayley Lincoln 0 5 6 A. Sorana Morrissy 2 5 6 Michael D. Taylor 2 5 6 Keiko Akagi 3 5 6 Cameron W. Brennan 1 5 6 Fausto J. Rodriguez 4 5 6 Lara S. Collier * 0 5 6 0 School of Pharmacy and University of Wisconsin Carbone Cancer Center, University of Wisconsin , Madison, Madison, WI , United States of America, 1 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center , New York, New York , United States of America, 2 Division of Neurosurgery, Arthur & Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children , Toronto, ON , Canada, 3 Comprehensive Cancer Center, The Ohio State University , Columbus, OH , United States of America, 4 Department of Pathology, Division of Neuropathology, Johns Hopkins University , Baltimore, MD , United States of America 5 Funding: This work was supported by R21- NS65352 and R01-NS085364 from the National Institute of Health (NIH) (LSC) and a grant from the Goldhirsh foundation (LSC); the Ohio Supercomputer Center PAS0425 (KA); the Leon Levy Foundation (CWB, Leon Levy Scholar) and NIH Grant P01-CA95616 (CWB). The UWCCC experimental pathology core is supported by NIH Cancer Center Support Grant (CCSG) P30- CA014520 and provided histopathology services. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 6 Editor: Andrew C. Wilber, Southern Illinois University School of Medicine , United States of America Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma. - High-grade gliomas are aggressive and invasive primary brain tumors with limited treatment options and a poor prognosis. The debilitating nature of the illness and poor results of therapy led to high-throughput genomic studies of human highgrade gliomas by The Cancer Genome Atlas (TCGA) and others [1, 2]. Genetic studies in mouse models serve as a complementary approach to human tumor genomic efforts to identify driver genes involved in glioma formation. Our previous studies [3, 4] demonstrated that the Sleeping Beauty (SB) transposon technology is capable of generating gliomas due to insertional mutagenesis of glioma genes. In these studies T2/onc transposons were mobilized from low-copy (LC, approximately 25 copies) concatemers throughout the body due to expression of SB transposase from the ROSA26 locus (Rosa26-SB11). Although approximately 90% of Rosa26-SB11; T2/onc LC mice developed leukemia, 14% of these mice harbored gliomas, primarily anaplastic astrocytomas. Tumorpredisposed genetic backgrounds increased glioma formation in mice with mobilizing transposons. Cloning insertions from these SB-induced gliomas identified Sfi1, Csf1, Mkln1, Vps13a and Fli1 as common insertion sites (CISs) which are chromosomal regions that are insertionally mutated in more tumors than would be expected by random chance and represent candidate glioma genes [4]. In order to extend these studies to generate an immunocompetent, autochthonous mouse glioma model useful for glioma gene discovery, we generated mice in which the SB11 version of the transposase is expressed from the human Glial Fibrillary Acidic Protein promoter (GFAP-SB11). We found that mobilizing T2 transposons with GFAP-SB11 on an otherwise wild-type background did not promote gliomas, and very rare gliomas were observed when transposons were mobilized on a p19Arf+/2 cancer predisposed background. In order to identify additional candidate glioma genes, we studied additional mice undergoing whole-body mutagenesis and identified new gliomas from these mice. Cloning of insertions from these tumors identified additional candidate glioma genes. Ethics Statement Mouse work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and was performed under the review and approval of the University of Wisconsin-Madison Institutional Animal Care And Use Committee. Animal condition was monitored daily by animal care staff and at least four times a week by an author who was blinded to genotype. Mice were euthanized by CO2 asphyxiation following AVMA Guidelines for the Euthanasia of Animals when any of the following humane endpoints were met: a body condition score of 2 or below [5], hunching behavior, lethargy, inappetence, failure to groom, progressive ataxia, growth retardation, hydrocephalus, seizure, or paralysis. All efforts were made to minimize suffering. Animals were housed with standard housing and husbandry conditions under specific pathogen free conditions. Animals received standard chow and water ad libitum. The only procedures that mice underwent were a tail clipping to provide sufficient genomic DNA for genotyping and an ear notch necessary to distinguish animals from each other. These procedures were carried out prior to weaning. T2/ onc2 high-copy (on chromosome 4), T2/onc low copy (lines 68 and 76), Rosa26SB11, p19Arf2/2, Blm2/2 and Csf1op/op (hereafter referred to as Csf12/2) mice have been previously described [610]. In addition, mice harboring a version of T2/onc with translational start sequences engineered into the MSCV LTR (T2/ oncATG) [4] were also utilized for some crosses to Rosa26-SB11. To generate mice expressing transposase in the GFAP compartment, the pGFAP-SB11 plasmid was constructed by excising SB11 from pCMV-SB11 by SacII digest and cloning it into pGFAP-Nrf2 [11, 12] that had the Nrf2 gene removed by SacII digest. The pGFAP-SB11 plasmid DNA was linearized with SphI and NdeI, and the 3.9 kb GFAP-SB11 transgene was used for pronuclear injections performed by the UWMadison Transgenic Animal Facility. Transgenic GFAP-SB11 mice were generated on the FVB/N genetic background. Potential (...truncated)