<i>Sleeping Beauty</i> Mouse Models Identify Candidate Genes Involved in Gliomagenesis

<div><p>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 <i>Sleeping Beauty</i> transposons in mice ubiquitously throughout the body from the <i>Rosa26</i> 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 <i>p19Arf</i> heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the <i>Rosa26</i> 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.</p></div

GFAP-SB11 transgenics express functional transposase in a subset of GFAP⁺ cells.

<p>Two lines (A and B) were established and used for these experiments. SB = SB transposase, T2 = T2/onc, LV = lateral ventricle. A) Immunofluorescence for GFAP (red) and SB (green). Nuclei are stained with DAPI. Arrows indicate examples of SB⁺ GFAP⁺ cells while asterisks indicate examples of SB⁻ GFAP⁺ cells. Scale bars are 20 µm. B) PCR based excision assay showing that transposons have mobilized in the brains of SB⁺T2⁺ but not SB⁻T2⁺ or SB⁺T2⁻ mice from each line. A control PCR demonstrates that genomic DNA is present for all samples.</p

FLI1 is expressed in a subset of cells in a glioma with a <i>Fli1</i> insertion.

<p>Immunoreactivity for FLI1 is in brown and immunoreactivity for IBA1 is in green. Nuclei are counterstained blue. A) 40× image of the dorsal third ventricular region in a control mouse without mobilizing transposons. B) 100× image of the boxed area in A. Asterisk indicates a FLI1 immunoreactive cell with morphologic features of a red blood cell. C) 40× image of the dorsal third ventricular region surrounded by tumor in AR151. D) 100× image of the boxed area in C. Arrowhead points to a nucleus that is negative for FLI1 and an arrow indicates an example of strong nuclear FLI1 staining. E) 100× image of tumor in AR151 that is distant from the ventricle. F) A 40× image of secondary only controls is shown for comparison to verify specific primary antibody staining. Scale bars = 50 µm.</p

CISs and gCISs identified in SB-gliomas.

a<p>Abbreviations used: GB = GenBank, N/A = not applicable, p value = p value for gCIS.</p><p>CISs and gCISs identified in SB-gliomas.</p

Genotypes and phenotypes of analyzed brains from Rosa26-SB11 crosses.

a<p>Abbreviations used: T2LC = T2/onc LC, T2ATG = T2/oncATG, p19 = p19Arf, wt = wild-type, AA = anaplastic astrocytoma, GBM = glioblastoma, PNET = primitive neuroectodermal tumor, DD = differential diagnosis.</p><p>Genotypes and phenotypes of analyzed brains from Rosa26-SB11 crosses.</p

A Dominantly Acting Murine Allele of <em>Mcm4</em> Causes Chromosomal Abnormalities and Promotes Tumorigenesis

<div><p>Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called <em>Spontaneous dominant leukemia</em> (<em>Sdl</em>). <em>Sdl</em> heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. <em>Sdl</em> mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from <em>Sdl</em> mice harbor small amplifications and deletions, including activating deletions at the <em>Notch1</em> locus. Using exome sequencing it was determined that <em>Sdl</em> mice harbor a spontaneously acquired mutation in <em>Mcm4</em> (<em>Mcm4^D573H</em>). MCM4 is part of the heterohexameric complex of MCM2–7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, <em>Sdl</em> mice possess normal levels of <em>Mcm</em>s, and there is no evidence for loss-of-heterozygosity at the <em>Mcm4</em> locus in <em>Sdl</em> leukemias. Studies in <em>Saccharomyces cerevisiae</em> indicate that the <em>Sdl</em> mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in <em>Sdl</em> mice result from the ability of MCM4^D573H to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.</p> </div

The <i>Sdl</i> mutation results in highly penetrant disease, which is primarily early-onset T-ALL.

<p>A) Kaplan-Meier curve of time to morbidity for <i>Sdl</i> mice. Known carriers of <i>Sdl</i> (harboring a C57Bl/6 haplotype at D16MIT131 and D16MIT4 on proximal Chr 16) are denoted by black squares, while sibling non-carriers are denoted by grey circles. p<0.0001. B–C) H&E staining showing that neoplastic cells fill hematopoietic organs (B) and also infiltrate the blood (vessel indicated with an asterisk) and the parenchyma (arrow) of other organs such as liver (C). B and C are 40× magnification, scale bar = 50 µM. D–G) Examples of flow cytometry analysis of lymphomas from four moribund <i>Sdl</i> mice. A full summary of flow-cytometry data is available as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003034#pgen.1003034.s007" target="_blank">Table S1</a>. Mice depicted in D–F succumbed to early onset-disease that is phenotypically T-ALL. Within these animals, there is evidence of both CD4/8 double positive (DP) disease as well as CD8 single positive (SP) disease. The mouse in G became moribund with late-onset disease (354 days of age) and the tumor cells do not express most T cell antigens (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003034#pgen.1003034.s007" target="_blank">Table S1</a>).</p

<i>Mcm4^D573H</i> acts in a dominant manner to promote tumorigenesis.

<p>WT = wild-type C = carrier. A) <i>Mcm2–7</i> transcript levels are not decreased in <i>Sdl</i> carrier thymuses (striped bars) compared to wild-type thymuses (solid bars) as analyzed by qRT-PCR. Values for wild-type thymus are normalized to 1. N = 3 for wild-type, 6 for carrier. Error bars represent standard deviation. There is a trend toward increased expression of <i>Mcm3</i> and <i>Mcm5</i> in <i>Sdl</i> carrier thymuses compared to wild-type thymuses (p = 0.07 and 0.09, respectively); all other p values >.2. B) Western analysis on total thymus protein extract as well as purified chromatin bound (c.b.) fractions indicate that <i>Sdl</i> carrier thymuses harbor similar levels of MCM2 and 4 proteins as do wild-type thymuses. TUBULIN and Ponceau S membrane staining were utilized to demonstrate equal loading for whole cell lysates and chromatin bound fractions, respectively. C) Sanger sequencing traces of RT-PCR products demonstrate that both wild-type (G) and mutant (C) <i>Mcm4</i> alleles are expressed in <i>Sdl</i> tumors and tumor-derived cell lines. RT-PCR products from 21-day-old wild-type and <i>Sdl</i> carrier thymuses are shown for reference. Arrow indicates dual G/C peak, asterisk indicates wild-type G peak.</p

Array CGH profiles of thymic tumors from three <i>Sdl</i> mice.

<p>Probes with copy number gains and losses in tumors compared to reference are shown in green and red, respectively.</p

Enriched Gene Ontology (GO) Terms between 21-day-old wild-type and carrier thymus.

<p>Enriched Gene Ontology (GO) Terms between 21-day-old wild-type and carrier thymus.</p