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>S. cerevisiae mcm4</i> engineered with the <i>Sdl</i> mutation at the equivalent residue (D632H) generates a non-biologically active helicase.

<p>A) Examples of genetic complementation tests of a <i>mcm4</i> deletion haploid strain in which viability is maintained by an URA3-<i>mcm4</i> plasmid. This strain was transformed with TRP1 plasmids expressing <i>mcm4^Sdl</i> mutation (S), <i>mcm4</i> wild-type (W) or empty TRP1 vector (V). A) Growth on permissive conditions (−TRP) demonstrates that all colonies analyzed harbor the expected TRP1 plasmids. B) Growth under restrictive conditions (−TRP+ FOA) occurs only if viability can be maintained by the allele on the TRP1 plasmid. All <i>mcm4</i> wild-type colonies grew under restrictive conditions and empty vector colonies do not, as expected. A fraction of colonies expressing <i>mcm4^Sdl</i> mutation grew under restrictive conditions. C) A restriction fragment polymorphism was utilized to distinguish <i>mcm4^Sdl</i> from wild-type (WT) <i>mcm4</i> sequences in the yeast strains described above. One <i>mcm4</i> wild-type (W) colony and 10 <i>mcm4^Sdl</i> (S) colonies are shown. All freshly isolated <i>mcm4^Sdl</i> colonies grown under permissive conditions (−URA −TRP) harbor both <i>mcm4^Sdl</i> and <i>mcm4</i> wild-type sequences due to the presence of TRP1-<i>mcm4^Sdl</i> and URA3-<i>mcm4</i> plasmids. All <i>mcm4^Sdl</i> colonies that grew under restrictive conditions lost <i>mcm4^Sdl</i> sequences, indicating that growth occurred due to a reversion or gene conversion event involving <i>mcm4^Sdl</i> sequences on the TRP1 plasmid and not due to the ability of <i>mcm4^Sdl</i> to complement the <i>mcm4</i> genomic deletion.</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

T cell development in wild-type and <i>Sdl</i> carrier mice.

1<p>Abbreviations used: WT = wild-type, C = Sdl carrier, SP = single positive, DP = double positive, DN = double negative.</p>2<p>t-test p value <0.064.</p>3<p>DN cells are defined as negative for all lineage markers (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003034#s4" target="_blank">Materials and Methods</a>).</p>4<p>t-test p value <0.014.</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

<i>Notch1</i> activation due to intragenic deletions occurs during leukemogenesis in <i>Sdl</i> mice.

<p>WT = wild-type, C = carrier <i>Sdl</i> thymus, T = tumor. Error bars represent standard deviation. For all qRT-PCR data, expression in wild-type thymus was normalized to 1. A) qRT-PCR detects higher levels of expression of the <i>Notch1</i> targets <i>Myc</i> and <i>Hes1</i> in <i>Sdl</i> thymic tumors compared to wild-type thymus or carrier thymus. N = 3 per group. p<0.001 for comparisons of wild-type thymus or carrier thymus to tumors. B) qRT-PCR results for querying expression levels of individual <i>Notch1</i> exon/exon boundaries indicated as well as exon 34 for three wild-type thymuses and five <i>Sdl</i> tumors. All q-PCRs were performed in triplicate. C) RT-PCR using a forward primer in exon 1 and a reverse primer in exon 30 detects abnormal <i>Notch1</i> transcripts in 11 of 15 tumors (T) from <i>Sdl</i> mice but not in wild-type thymus (WT) or thymus from <i>Sdl</i> carrier mice (C) (top panel). An additional tumor (asterisk) was weakly positive. A no-RNA control (water, W) is also shown. RT-PCR for <i>Gapdh</i> was used to verify the presence of cDNA (bottom panel). D) Sequences surrounding three genomic breakpoints in <i>Notch1</i> cloned from <i>Sdl</i> tumors. 5′ introns are in regular font, 3′ introns are in bold font. Microhomology is underlined.</p