Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

et al. (2014) Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice. PLoS ONE 9(5): e97280. doi:10.1371/journal.pone.0097280 Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice Raha A. Been 0 Michael A. Linden 0 Courtney J. Hager 0 Krista J. DeCoursin 0 Juan E. Abrahante 0 Sean R. Landman 0 Michael Steinbach 0 Aaron L. Sarver 0 David A. Largaespada 0 Timothy K. Starr 0 Andrew C. Wilber, Southern Illinois University School of Medicine, United States of America 0 1 Masonic Cancer Center, University of Minnesota , Minneapolis , Minnesota, United States of America, 2 College of Veterinary Medicine, University of Minnesota , St. Paul , Minnesota, United States of America, 3 Department of Comparative and Molecular Biosciences, University of Minnesota , St. Paul , Minnesota, United States of America , 4 Obstetrics, Gynecology, and Women's Health , University of Minnesota , Minneapolis , Minnesota, United States of America, 5 Department of Laboratory Medicine and Pathology, University of Minnesota , Minneapolis , Minnesota, United States of America, 6 Department of Genetics, Cell Biology, and Development, University of Minnesota , Minneapolis , Minnesota, United States of America, 7 Department of Computer Science and Engineering, University of Minnesota , Minneapolis, Minnesota , United States of America Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients. - Funding: RAB and DAL were supported by grants from the NCI (R01 CA113636) and the Leukemia and Lymphoma Society (LLS 7019-04). SL was supported by NIH Grant T32EB008389, Integrative Training in Neuroimaging, and by a University of Minnesota Interdisciplinary Doctoral Fellowship. JEA was supported by an SP3 grant from the Masonic Cancer Center. TKS was supported by a grant from the American Cancer Society (PF-06-282-01-MGO), the NIH (4R00CA151672-02), Masonic Cancer Center shared resources (NIH P30 CA077598) and generous assistance from the Masonic Cancer Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Histiocytic sarcoma (HS) is classified as a neoplastic proliferation with features of histiocytes/macrophages[1]. HS has also been called true histiocytic lymphoma or malignant histiocytosis, but these terms have been discontinued. Before 1990, the majority of patients diagnosed with HS were misdiagnosed due to a lack of antibodies specific for the histiocytic lineage. Retrospective analysis indicated the majority of these patients actually had Bor T-cell lymphomas[25]. Case studies have demonstrated that HS can occur in isolation or in the context of other hematological malignancies, such as B-cell lymphoma, to which the HS is sometimes clonally related[4]. HS may thus, in some cases, develop via trans-differentiation from a malignant, or premalignant, lymphoid neoplasm. HS is rare, with an incidence far less common than the non-Hodgkin lymphomas[1,6]. Typically, patients present with advanced clinical disease and have a poor prognosis[1,4,5]. Since the genetic etiology of HS is largely unknown, HS is difficult to manage clinically and there is no standard therapy for patients with HS. Currently, no precursor lesions or etiologic agents have been described for human HS[7]. Two cytogenetic case studies identified gains in chromosome 8 in human HS[8,9], implicating MYC as a HS oncogene. Animal models have identified possible driver genetic lesions. ArrayCGH performed on over 100 canine HS samples revealed an average of 30 copy number alterations per tumor[10], while a genome wide association study in Bernese Mountain Dogs identified a strong association between HS and the MTAP-CDKN2A locus[11]. Pten and Ink4aARF are also implicated, as compound heterozygous mice develop HS and 60% of human HS examined for protein expression show a loss of PTEN, p16INK4A, or p14ARF[12]. Several other genetic mouse models have produced HS including Dok1/Dok2/Dok3 triple knockout animals[13], Cyp1b1 knockout mice[14], p21 knockout mice[15], and p19ARF/Bax mutant mice[16]. In addition, 50% of Cdkn2a deficient mice infected with Moloney murine leukemia virus developed HS, which was frequently accompanied by lymphoma[17]. To identify genetic drivers of HS we performed an unbiased forward genetic screen in mice using the Sleeping Beauty (SB) transposon as an insertional mutagen[1820]. SB is capable of both activating proto-oncogenes and inactivating tumor suppressor genes and has been used to identify genetic drivers in a variety of cancers[2132]. In this study we activated SB mutagenesis using the Lysozyme2 (Lyz2) promoter in a cohort of mice resulting in early mortality and a large percentage of mice developing HS. Analysis of transposon CISs identified 28 genes, including 2 miRNAs associated with HS. Several of these genes are known oncogenes and tumor suppressors including Nf1, Pten, Myc and Fli1, while many others have not been directly associated with cancer and could be potential targets for therapy. Methods and Materials Ethics Statement All mice were bred, cared for and euthanized in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee (Protocol # 0901A56501). Transgenic Mice Lyz-Cre mice were obtained from Jackson Laboratories (Strain name: B6.129P2-Lyz2tm1(cre)Ifo/J, Cat # 004781)[33]. These mice were created using a knock-in allele that has a nuclear localized Cre recombinase cDNA inserted into the first coding ATG of the Lyz2 gene. This allele abolishes endogenous Lyz2 gene function and places NLS-Cre expression under the control of the endogenous Lyz2 (...truncated)