Generation of a Mouse Model of Atypical Teratoid/Rhabdoid Tumor of the Central Nervous System through Combined Deletion of Snf5 and p53

Malignant rhabdoid tumors (MRT) are aggressive, poorly differentiated pediatric cancers, characterized by the presence of germline and/or somatic mutations in Snf5/Ini1/Baf47/Smarcb1, which functions as a component of the SWI/SNF core chromatin-remodeling complex (1). Tumors arise frequently in kidney and soft tissues and less frequently in the central nervous system (CNS). In the CNS, neoplastic lesions, referred to as atypical teratoid/rhabdoid tumors (AT/RT), occur primarily in early childhood with a median age of onset of 11 months (2). Most children die from disease within 1 year of diagnosis, despite use of intensive therapy.

Snf5/Ini1 is a nuclear protein that is constitutively expressed in normal cells but absent in AT/RT (3). The majority of rhabdoid tumors exhibit LOH at the Snf5/Ini1 locus and harbor recurrent biallelic alterations (deletions and point mutations) irrespective of the tissue of origin (2, 4). While several studies demonstrate a role for Snf5 in the regulation of cyclin D1, p16Ink4a, and pRb, through the ATP-dependent chromatin-remodeling SWI/SNF complex, the mechanism responsible for oncogenesis remains unclear (5). Ink4 and Arf signaling have been suggested to be disrupted in AT/RT (6), and reintroduction of Snf5/Ini1 into rhabdoid tumor cells causes G₀–G₁ arrest and senescence by direct repression of cyclin D1 and activation of p16Ink4a (7–9). It has been proposed that Snf5/Ini1 activates the mitotic spindle checkpoint through the p16–cyclinD1/Cdk4–pRb–E2F pathway (10). The IFN and hedgehog (Hh) signaling pathways have also been proposed to be affected by Snf5/Ini1 (11, 12), and it has been suggested that oncogenesis in the absence of Snf5 requires residual activity of BRG1-containing SWI/SNF complexes (13).

Alterations in Snf5/Ini1 have also been reported in familial and sporadic schwannomatosis (14, 15) and in gastrointestinal stromal tumors (16). Furthermore, loss of Snf5/Ini1 has been documented in renal medullary carcinoma, possibly as a consequence of epigenetic silencing (17). Taken together, these data imply that Snf5 is a tumor suppressor gene involved in oncogenic transformation of cells present in a broad range of developing tissues.

In mice, homozygous deletion of Snf5/Ini1 results in early embryonic lethality, whereas heterozygous loss, similar to the situation in humans, predisposes to development of aggressive sarcomas (18–20). Conditional inactivation of Snf5 in mice results in profound cancer susceptibility, with all animals developing tumors at a median age of 11 weeks (21). These lesions exhibit many features of rhabdoid tumors, including the complete absence of Snf5/Ini1 expression. Homozygous or heterozygous deletion of p53 in Snf5 heterozygous mice accelerates the appearance of MRTs (22, 23). However, to date, no brain tumors have been reported in mice carrying mutations in Snf5.

To investigate the role of Snf5 in the developing CNS, we targeted Snf5 and p53 mutations to neuronal progenitor cells using Cre-lox technology. Ablation of Snf5 in nestin-positive neural progenitor cells resulted in embryonic lethality, which was not rescued by the absence of p53. In contrast, initially only modest neuroanatomic defects were observed in Snf5^Flox/GFAP-Cre mice, indicating a strong lineage-dependent effect of Snf5 ablation. However, in the absence of p53, Snf5^F/GFAP-Cre mice exhibited neurodegeneration and defects in granule neuron migration, ataxia, and seizure activity. These phenotypes appear to arise as a consequence of the loss of oligodendrocytes throughout the developing brain. In addition, all adult Snf5^F/F/p53^lox/lox/GFAP-Cre mice examined, after the age of 4 weeks, exhibited highly aggressive brain tumors displaying many hallmarks of CNS AT/RT, including loss of Snf5 expression.

GFAP-Cre (GFAP, glial fibrillary acidic protein) transgenic mice, a gift from David H. Gutmann (Washington University School of Medicine, St. Louis, MO), have been described previously (24, 25) to drive expression of Cre recombinase specifically in the developing nervous system of the mouse. To obtain Snf5 conditional loss in the nervous system of the mouse, Snf5^F/F mice (21) provided by Charles Roberts (Dana-Farber Cancer Institute and Children's Hospital, Harvard Medical School, Boston, MA) were crossed with GFAP-Cre mice for two generations to generate Snf5^F/F/GFAP-cre mice, as well as littermate controls. Inactivation of p53 was achieved using p53^lox/lox mice, obtained from the National Cancer Institute (Bethesda, MD), to generate Snf5^F/F/p53^L/L/GFAP-Cre animals. Genotyping of Snf5^F, p53^L, and Cre are described in Supplementary Data. Ptc1^+/− mice obtained from Dr. Mathew Scott (Stanford University, Stanford, CA) were maintained on a mixed C57Bl/6 and 129Sv strain background and were crossed with p53^+/− mice (Jackson Laboratory) to generate Ptc1^+/− p53^+/− and Ptc^+/− p53^−/− mice. Genotyping of Ptc1 and p53 was performed as previously described (26, 27).

Animals were transcardially perfused with 1× PBS followed by 4% paraformaldehyde in PBS. For some experiments, mice were sacrificed by cervical dislocation after intraperitoneal injection with ketamine (100–300 mg/kg) and xylazine (16–48 mg/kg). Following dissection, brain tissues were transferred to PBS and subsequently embedded in paraffin and sectioned at a thickness of 5 μm. Every tenth section was stained with hematoxylin and eosin (H&E), and sections from similar anatomical planes were chosen for histologic and immunohistochemical analyses. Histopathologic analysis was performed in a blinded manner. Immunohistochemical and double-label immunofluorescent (IF) staining were performed on formalin-fixed, paraffin-embedded brain sections. Procedure details and antibodies are described in Supplementary Data.

All electroencephalography (EEG) studies were performed on Snf5/p53/GFAP-Cre mice. Mice carrying the Cre alleles were used as controls. The methods for implantation and recording have been previously published (28, 29) and details are presented in Supplementary Data. EEG tracings were visually reviewed for the presence of electrographic seizures. If a seizure was suspected, the video was reviewed to ensure the electrographic change was associated with a seizure and not associated with movements, such as scratching, which could produce an artifact. The EEG was also qualitatively reviewed to characterize the background activity, including the amplitude, frequencies, and the presence of normal transients (e.g., theta in hippocampal electrodes during exploration), and interictal epilpetiform abnormalities such as spikes. These patterns were compared in all groups monitored.

Total RNA was isolated from mouse AT/RT derived from Snf5^F/F/p53^L/L/GFAP-Cre mice (n = 5) and from medulloblastoma derived from Ptc1^+/− p53^+/− (n = 2) and Ptc1^+/− p53^−/− (n = 3) mice using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Two hundred nanograms of total RNA was amplified and converted to biotinylated cRNA using the MessageAmp Premier Ampification Kit (Revision G, Life Technologies). Ten micrograms of fragmented cRNA was used for each Affymetrix Mouse Genome 430A 2.0 microarray, with hybridization, washing, staining, and scanning performed according to the standard protocol (Expression Analysis Technical Manual, Revision 3, Affymetrix). Raw data from the GeneChip 3000 7G scanner, converted to CEL intensity files using Affymetrix Command Console version 4.1.2, were used for subsequent analyses. Detailed qRT-PCR and gene expression of human and mouse data sets are described in Supplementary Data. All data have been deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=mropwgcsphczhat&acc=GSE68627).

The nestin-Cre transgene (30) was introduced into mice to ablate Snf5 and p53, individually and together, from the developing nervous system. Loss of Snf5 alone in neuronal progenitor cells resulted in embryonic lethality, as no live pups homozygous for Snf5 loss (0/79) were obtained from heterozygous intercrosses of Snf5^+/F nestin-Cre mice. In contrast, heterozygous Snf5^+/F nestin-Cre mice (n = 85) were grossly normal and fertile and did not exhibit any signs of disease up to one and half years of age. Very small litter sizes (average of 5) were obtained from these crosses, indicating that most embryos died in utero. To rescue the suspected lethality, we crossed Snf5^+/F nestin-Cre mice with p53 Lox mice, however, even in the absence of p53, we did not obtain any live Snf5^F/F nestin-Cre pups (0 of 151). Thus, Snf5 expression in nestin-expressing neural progenitor cells is essential for completion of embryonic development.

To target additional mouse neural stem/progenitor cell populations, we used the human GFAP promoter to program Cre expression (GFAP-Cre; refs. 24, 25) in Snf5^F mice. This promoter drives Cre expression in neural progenitor cells and a small subset of astrocytes (31). Snf5^+/F/GFAP-Cre and Snf5^F/F/GFAP-Cre mice were grossly normal and fertile. However, histologic analysis of brain sections revealed evidence of a degenerative phenotype in all Snf5^F/F/GFAP-Cre mice examined (Supplementary Fig. S1). The extent of the damage was variable and it progressed with age. The lesions were prominent in the white matter of the corpus callosum with vacuolization, spongy changes, cystic-like breakdown, and destruction of tissue. The posterior portion of the corpus callosum (splenium) was more affected than the anterior portion (genu). Loss of axons and myelin were also detected in the mutant brains that were stained with myelin basic protein (MBP) and proteolipid protein (PLP) antibodies, whereas both proteins were abundant in control animals. However, the cerebral cortex, although thinner, and the pyramidal cells of the hippocampus were mostly well preserved. The cerebellum appeared grossly normal, although there was a variable, mild defect in granule neuron migration and disorganization of the Purkinje cell layer. We also found slightly reduced levels of cells expressing oligodendrocyte lineage transcription factor 2 (Olig2) in the hippocampal region of the mutant brain (Supplementary Fig. S1C). In the control brain, we counted 388 cells in total, of which 229 cells were positive for Olig2, whereas in the mutant brain, 174 total cells were counted, of which 89 cells were positive for Olig2. This indicates that on a unit basis, there is a reduction in the total number of cells from 194 (control) to 58 (mutant), whereas the number of Olig2-positive cells decreased from 150 (control) to 30 (mutant). These data suggest that there is 80% reduction in the percentage of Olig2-positive cells in the area examined in the mutant brain. In addition, there was decreased expression of the specific neuronal marker β-III-tubulin in the mutant brains (Supplementary Fig. S1D).

Over the course of more than 1 year, no brain tumors developed in any Snf5^+/F/GFAP-Cre or Snf5^F/F/GFAP-Cre mice. We performed histologic analyses of a total of 30 brains from Snf5^+/F/GFAP-Cre and Snf5^F/F/GFAP-Cre mice, from ages 2 months to 1 year, and did not detect any evidence of neoplastic growth. Because p53 loss or reduced p53 expression have been reported to accelerate peripheral tumors in mice heterozygous for Snf5 (19, 22, 23), we investigated whether p53 ablation would promote brain tumor formation by crossing Snf5^F/F/GFAP-Cre mice with p53 Lox mice.

Mice heterozygous for Snf5 (Snf5^+/F/p53^+/L/GFAP-Cre, n = 54 and Snf5^+/F/p53^L/L/GFAP-Cre, n = 110) appeared grossly normal and fertile, although they displayed excessive scratching behavior, resulting in dermatitis around the neck, earlobes, and the flank region. In contrast, Snf5^F/F/p53^+/L/GFAP-Cre mice showed similar phenotypic and pathohistologic features to those seen in Snf5^F/F/GFAP-Cre animals, including extreme lesions in the white matter of the corpus callosum and cingulum, reduced levels of expression of MBP, PLP, Olig2, and β-III-tubulin. However, there was no evidence of neoplastic growth. Double homozygous mice (Snf5^F/F/p53^L/L/GFAP-Cre, n = 240) were much smaller than their heterozygous littermates (Fig. 1A, left), with bodyweight reduced by approximately 40% (Fig. 1A, right), and they required water gel for survival. In addition, they displayed severe ataxia with dystonia and seizures. Many mice also appeared quite lethargic with hind limb paralysis. Furthermore, they exhibited a more severe version of the scratching behavior seen in Snf5^+/F/p53^L/L/GFAP-Cre and Snf5^F/F/p53^+/L/GFAP-Cre mice.

Snf5^F/F/p53^L/L/GFAP-Cre mice exhibited a strong neurological phenotype, tumbling repeatedly during attempted locomotion. Motor coordination problems, manifest as tremors, dystonia, and abnormal posture of the hind limbs, became evident from around 3 weeks of age to adulthood. We used a foot painting assay to trace the walking gait of adult mice. Two colors of nontoxic water-based paint were used to mark the bottom of the forepaws (blue) and hindpaws (red), and the mice were placed onto white paper to trace their footprints. Wild-type animals displayed steady, evenly measured strides, with hind- and forepaw positioning resulting in overlapping footprints and a uniform gait width (Fig. 1B, left). In contrast, Snf5^F/F/p53^L/L/GFAP-Cre mice had a nonuniform gait and exhibited circling behavior. The traces indicated uneven stride length and width, with smeared nonoverlapping footprints, indicating dragging rather than precise positioning of the hindpaws and an inability to keep the hindquarters upright (Fig. 1B, right). Also, Snf5^F/F/p53^L/L/GFAP-Cre mice frequently fell over, and they repeatedly lifted and replaced the same paw in various positions during each stride. Ataxia, combined with dystonia and tremors, persisted into adulthood. Footprint tracings from single mutant floxed (Snf5^F/F/GFAP-Cre) or double heterozygous mutant floxed mice (Snf5^+/F/p53^+/L/GFAP-Cre Snf5^+/F/p53^L/L/GFAP-Cre) appeared quite normal, indicating a straight path with regular alternating strides, and they were never observed to lose balance during locomotion. These results demonstrate that loss of Snf5 results in ataxia and they imply that Snf5 is required for normal functioning of the cerebellum.

Observations of Snf5^F/F/p53^L/L/GFAP-Cre mice suggested possible seizure activity. To investigate this possibility, we performed long-term video EEG analysis of Snf5^F/F/p53^L/L/GFAP-Cre and wild-type mice at 3 months of age. EEG measures electrical activity in the brain. The three mutant mice examined showed lower voltage and slower background EEG activity than wild-type mice [Fig. 2; note the tracings are displayed at the same gain (mV/mm) making the control look higher than the mutant.] The low voltage tracing included intermittent runs of sharp waves (bracketed arrows in Fig. 2A, lower tracings) that sometimes evolved into clear seizures (bracketed arrows in Fig. 2B, upper tracings). The Snf5^F/F/p53^L/L/GFAP-Cre mice also exhibited intermittent electrographic seizures (Fig. 2B) associated either with forelimb clonus, evolving into body jerking, and wild running (stage 5 seizure; ref. 32), or more commonly arrest of activity during the ictal EEG discharges. All three of the recorded mice developed spontaneous seizures. The EEG changes were consistently composed of a buildup of rhythmic spiking in both the cortex and hippocampus (arrows in Fig. 2B).

Snf5^F/F/p53^L/L/GFAP-Cre mice were also observed to have intermittent episodes of tail extension and dystonic posturing of the body, frequently in response to handling. To determine whether these movements were seizures, during the EEG recordings, the mice were manipulated to induce the tail extension and dystonic posturing. For all three mutant mice recorded, we observed these behaviors after the mice were manipulated and while the animals were spontaneously walking around the recording cage. At no time, was there an associated seizure like electrographic discharge, strongly suggesting that these movements were not related to cortical or hippocampal seizure activity. Together, these data indicate that the Snf5^F/F/p53^L/L/GFAP-Cre mice had both spontaneous electrographic seizures and paroxysmal events of odd positioning and abnormal gait that did not have an electrographic correlate.

Histologic analysis of adult Snf5^F/F/p53^L/L/GFAP-Cre mouse brain revealed profound defects in the white matter, corpus callosum, hippocampus, and cerebellum compared with Snf5^F/F/GFAP-Cre animals (Fig. 3). Immunostaining using Ini1/Baf47 antibody demonstrated normal, nuclear expression in most cell types in control brain sections throughout the cortex, hippocampus, and cerebellum (Fig. 3B and D). In contrast, loss of Snf5 expression was evident throughout Snf5^F/F/p53^L/L/GFAP-Cre brains in several specific cell populations. For example, loss of Snf5 expression was observed in approximately 20% of cells in the CA1 hippocampal region of mutant mice (52 of 280) compared with control mice (0 of 511) (Fig. 3B). Snf5 loss was evident in rare scattered cells throughout the thinner cerebral cortex of mutant mice (Fig. 3B). However, the most prevalent site of Snf5 loss was in the cerebellum in both white matter and some populations of cells in the disrupted granule cell layer (Fig. 3D, red arrowheads). Thus, Snf5^F/F/p53^L/L/GFAP-Cre mice showed Snf5 inactivation in several subsets of brain cells. Cell counting also revealed reduced total cell numbers in specific regions of the mutant mouse brains. The amount of cell loss increased over time and, ultimately, there was complete loss of tissue in regions of white matter.

Analysis of the cerebellar organization of Snf5^F/F/p53^L/L/GFAP-Cre mice revealed defects in cytoarchitecture and altered granule neuron migration (Fig. 3C and D). Some granule neurons failed to migrate out of the external germinal layer, others remained trapped in the molecular layer and the boundary between the internal granule layer and the molecular layer remained diffuse. Remarkably, Snf5 was expressed in the majority of ectopic granule neurons, suggesting that the defect in migration may be non–cell-autonomous. In addition, there was a small patch of neoplastic, proliferating cells involving the subpial molecular region, located superficially at the interface of two cerebellar folia, representing an early neoplastic lesion. As expected, these neoplastic cells demonstrated a loss of nuclear expression of Ini1/Baf47 (Fig. 3D, blue arrows). At higher magnification, large atypical cells, some with rhabdoid features and many mitoses, supporting a highly proliferative activity, are also present (Supplementary Fig. S2).

Compared with control cerebellum, white matter fiber tracks appeared to be much reduced in the mutant mice. Many of the cell nuclei present in these regions were negative for Snf5. To determine whether this was a developmental phenotype, we examined the brains of 3-week-old mice (Fig. 4). The defects observed in adult mice, including lesions in the corpus callosum, reduced cerebral cortex, loss of white matter, altered cerebellar granule neuron migration (Fig. 4A and B, yellow arrowheads), were all present at 3 weeks of age. However, in this case, there were many more cells present in the cerebellar white matter, and in the most superficial aspect of the cerebellar folia, that were negative for Snf5 staining (Fig. 4B, red arrows). Staining with GFAP antibodies revealed high levels of gliosis in the fiber tracks and in the molecular layer as well as in the remnants of the aberrant external germinal layer (Fig. 4C, red arrow and arrowhead, respectively). These findings imply that there is a progressive loss of cells and neuronal projections in the cerebellum of Snf5^F/F/p53^L/L/GFAP-Cre mice.

Because the most prevalent site of loss of Snf5 expression in the cerebellum was observed in fiber tracks, we hypothesized that these cells may be oligodendrocytes. Immunofluorescent histochemical analysis revealed a profound loss of cells positive for the oligodendrocyte markers MBP, PLP, and Olig2, in Snf5^F/F/p53^L/L/GFAP-Cre mice compared with control mice (Fig. 5). The prevalence of Snf5-negative oligodendrocytes dropped dramatically as the animals aged, indicating that these cells were lost. Loss of oligodendrocytes is associated with neurodegeneration and seizure activity (33, 34). In fact, ablation of mouse oligodendrocytes during the first 3 postnatal weeks causes a developmental phenotype, including abnormal cerebellar foliation, impaired neuronal migration, seizures, tremors, and retarded growth, that is remarkably similar to that of Snf5^F/F/p53^L/L/GFAP-Cre mice (33, 34). In contrast, the levels of NG2+ oligodendrocyte precursor cells were comparable in 3-week-old mutant mouse cerebellum and hippocampus compared with control mice (Supplementary Fig. S3A). Similar levels of NG2+ oligodendrocyte precursor cells were also seen in adult mutant mouse cerebellum and hippocampus compared with control mice (Supplementary Fig. S3B). In adult mutant mice, very few Olig2-positive/Baf47-negative remain in the cerebellum (Supplementary Fig. S4). These results imply that Snf5 plays a fundamental role in the generation and maintenance of oligodendrocytes after the NG2+ stage.

Several Snf5^F/F/p53^L/L/GFAP-Cre adult mice of various ages died suddenly, independently of the severity of their ataxic or seizure phenotypes. Histologic examination of brain tissues from these mice revealed the presence of high-grade, aggressive tumors, that infiltrated and obliterated the surrounding cerebellar folia (Fig. 6). Subpial and leptomeningeal spread as well as infiltration into the mesencephalon was also seen in some mice; however, all tumors appeared to arise from the cerebellum. Mice were examined when they showed the first signs of disease. However, the age of onset was distributed across a 12-month period. Histologically, the tumors displayed many of the hallmarks of AT/RT. The classical rhabdoid cells, with eccentrically placed nuclei, prominent nucleoli, and eosinophilic inclusion-like cytoplasm (Fig. 6B and C, arrows and arrowheads) were intermingled with primitive neuroectodermal tumor (PNET)-like small cells. In many cases, this PNET-like appearance was the predominant pattern. This is similar to human AT/RT, in which only a minority of neoplastic cells demonstrate typical rhabdoid features with the majority represented by primitive PNET-like small cells. In keeping with the immunohistochemical profile of AT/RT, all tumors examined (n = 36) demonstrated loss of nuclear Ini1/Baf47 staining. Positive staining was seen only in stromal endothelial cells (Fig. 6D; ref. 3). Strong reactivity for cytokeratin was seen in groups of cells, or single elements, with a maximum of up to 30% to 40% of the tumor cells staining positive, depending on the specific case (CK, Fig. 6E). The same variability, with patchy and focal staining, was observed with synaptophysin (SYN, Fig. 6F) and GFAP (Fig. 6G). Some tumors were negative for SYN, and the scattered cells reactive for GFAP were identified as reactive astrocytes rather than tumor cells. Neither human AT/RT (n = 17) nor our mouse tumors stained positive for NG2 (data not shown). In addition, Ki67, a nuclear marker of the cell cycle, demonstrated high reactivity (50%–60% and in some cases even up to 70–80%; Fig. 6H), and the apoptotic marker caspase-3 was also observed (not shown). The other widely used markers for AT/RT, smooth muscle actin and epithelial membrane antigen, do not work on mouse tissues.

Snf5^F/F/p53^L/L/GFAP-Cre mice developed brain tumors starting at around 1 month of age (n = 8) and they were observed in all adult mice examined (n = 58). Taking together the histologic appearance, immunohistochemical profile, and loss of Snf5 expression in tumors cells, we conclude that the tumors arising in Snf5^F/F/p53^L/L/GFAP-Cre mice should be considered as AT/RT.

Human medulloblastoma can be segregated into four major categories on the basis of gene expression patterns: Hedgehog (HH) subtype, Wnt subtype, group 3, and group 4 (35, 36). However, they all exhibit strikingly different gene expression profiles from AT/RT (37). We reanalyzed published gene expression microarray dataset of pediatric brain tumors to identify a gene expression profile that clearly distinguished human AT/RT from human medulloblastoma (37). We used this profile, choosing only genes that have clear murine orthologs, to compare tumors from Snf5^F/F/p53^L/L/GFAP-Cre mice with medulloblastoma from Ptc1^+/− mice. As illustrated in Fig. 7A–C, Snf5^F/F/p53^L/L/GFAP-Cre tumors are clearly very different from mouse medulloblastoma and the markers that distinguish human AT/RT from human medulloblastoma also distinguish the mouse tumors. We confirmed this gene expression array finding using a selection of specific marker genes in a qRT-PCR assay (Fig. 7D). The set of genes include 3 controls, 5 genes more highly expressed in human AT/RT, and 5 genes more highly expressed in human medulloblastoma. The data are very consistent with analysis of human brain tumors and clearly show that our mouse tumors closely resemble human AT/RT. Details of the analysis, including gene set enrichment analysis (GSEA), are presented in Supplementary Fig. S5.

Genetically engineered mouse (GEM) models have allowed significant advances to be made in the understanding of human cancer and, in the case of hedgehog pathway inhibitors, they allowed critical proof-of-concept studies that accurately predicted subsequent clinical responses (38, 39). Here, we report a new GEM, Snf5^F/F/p53^L/L/GFAP-Cre that provides a model for CNS AT/RT. While no mouse model can encompass the diversity of human cancer, Snf5^F/F/p53^L/L/GFAP-Cre mice recapitulate the histopathology, invasive phenotype, and aggressive growth of AT/RT brain tumors. In addition, gene expression analysis indicated that our mouse tumors share a gene expression profile with human AT/RT that distinguishes them from medulloblastoma. We hope that these mice, and tumor cells derived from the mice, propagated in culture or in allograft tumor transplants and can be used to develop new approaches for the treatment of this devastating pediatric disease.

Ablation of Snf5 using a nestin-Cre expression construct resulted in prenatal lethality. The cause of the lethality was not determined, but it was not rescued by the absence of p53. Complete ablation of Snf5 results in embryonic lethality around the time of implantation (18, 20) and targeted ablation at birth, using the inducible MX-Cre construct to achieve widespread excision in many tissues, with the exception of the brain, results in hematopoietic failure leading to death of most mice at 1 to 3 weeks of age (40). Thus, Snf5 seems to be essential for survival of neuronal lineages, as it is in the majority of cell types.

In the case of the GFAP-Cre construct, mice did survive, but they exhibited an unexpected progressive neurologic phenotype. Although the age of onset and severity of the phenotype varied, all mice examined displayed some histopathologic evidence of white matter degeneration, neuronal loss, and mild defects in cerebellar granule neuron migration. The defects in granule neuron migration appear to be non–cell-autonomous, as Snf5 was present in ectopic granule neurons. The human GFAP-Cre construct (Gfa2) used for these studies has been widely studied and shown to direct Cre-mediated recombination in astrocytes, oligodendroglia, ependymal cells, and a subset of neurons (24, 41, 42). Initial analysis indicated there was some loss of oligodendrocytes and neuronal projections.

Whole-exome sequencing of 32 human MRTs, including 20 CNS AT/RT, revealed only one p53 mutation that was predicted not to be detrimental, although this was not demonstrated (43). However, previous mutational analysis identified p53 mutations in 3 of 19 CNS AT/RTs nucleophosmin (6). In addition, the majority of CNS AT/RT express p53 and it was proposed that p53 is inhibited in these tumors by binding (44, 45). Thus, there is support for the notion that deregulation of p53 plays a role in AT/RT. Furthermore, loss of p53 accelerates tumor formation in mice with heterozygous mutations in Snf5 (19, 23), and it has been argued that reduced expression of p53 cooperates with loss of Snf5 to decrease the latency of tumors arising in sites observed infrequently in Snf5^+/− mice because Snf5 regulates a subset of p53 target genes (22, 46). Thus, there is strong evidence of cooperation between these two tumor suppressor pathways. In the case of the GFAP-Cre GEM, we observed a very high degree of cooperativity, as no tumors were observed in the presence of p53, and 100% of Snf5^F/F/p53^L/L/GFAP-Cre mice examined after 3 weeks of age harbored brain tumors with many hallmarks of AT/RT. Cooperativity extended to the neurologic phenotype observed in Snf5^F/F/GFAP-Cre mice, which was much more severe in Snf5^F/F/p53^L/L/GFAP-Cre mice. We did not detect any tumors or enhancement of the neurologic phenotype in Snf5^F/F/p53^+/L/GFAP-Cre mice; therefore, the basis of the cooperativity we observed may be different than that proposed to explain acceleration of peripheral MRTs (46). In general, p53 loss during tumor initiation has been suggested to promote tumorigenesis by blocking apoptosis. However, there was increased cell death in the oligodendrocyte lineage, and in some neuronal populations, in Snf5^F/F/p53^L/L/GFAP-Cre mice. While we do not yet know the cell of origin of AT/RT, these results imply that there are additional biologic consequences of the interactions between loss of Snf5 and p53 that may be cell-type–dependent.

The profound neurodegeneration phenotype observed in Snf5^F/F/p53^L/L/GFAP-Cre mice was quite unexpected. The phenotype seems to be initiated by a dramatic loss of oligodendrocytes, as it is very similar to the phenotype described in mice lacking oligodendrocytes (33, 34). At 3 weeks of age in Snf5^F/F/p53^L/L/GFAP-Cre mice, there are many oligodendrocytes that are negative for Snf5 immunohistochemistry. Subsequently, these cells were lost leading to a progressive degeneration of nerve fibers and a host of ancillary phenotypes, including seizure activity. Because some myelin-positive cells can be detected initially, it seems that Snf5 may be more critical for the maintenance rather than the generation of oligodendrocytes. Children with AT/RT suffer a broad range of neurological deficits, many of which have been ascribed to the invasive nature of the tumor or the side effects of therapy (47). However, it is also possible that germline, or somatic, alterations in Smarcb1, such as those seen in patients with Coffin–Siris syndrome, could also contribute to the neurodevelopmental phenotypes seen in these patients (48). Our studies imply that some of these effects may be a consequence of loss of oligodendrocytes.

The earliest tumors observed in mutant mice appeared to arise in the most superficial aspects of the cerebellar folia. The tumors are invasive and highly aggressive destroying surrounding cerebellar tissues as they grow. The comorbidity arising from the loss of oligodendrocytes begs the question of whether AT/RT are derived from progenitor cells in this lineage. Interestingly, germline or somatic loss of Snf5 is prevalent in schwannoma (15, 49). Furthermore, nPDGFA/NG2-expressing glial progenitor/stem cells can produce both oligodendrocytes and Schwann cells during repair of chemical CNS demyelination (50). Thus, it is not out of the question that rhabdoid tumors are derived from cells in these allied lineages of the central and peripheral nervous systems.

No potential conflicts of interest were disclosed.

Conception and design: J.M.Y. Ng, T. Curran

Development of methodology: J.M.Y. Ng, T. Curran

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.M.Y. Ng, D. Martinez, E.D. Marsh, E. Rappaport, M. Santi, T. Curran

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.M.Y. Ng, E.D. Marsh, Z. Zhang, E. Rappaport, M. Santi, T. Curran

Writing, review, and/or revision of the manuscript: J.M.Y. Ng, D. Martinez, E.D. Marsh, Z. Zhang, T. Curran

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.M.Y. Ng, T. Curran

Study supervision: J.M.Y. Ng, T. Curran

Other (conception and design of EEG-related experiment): E.D. Marsh

This work was supported by grants from the Children's Brain Tumor Foundation, the Brain Tumor Society, and the NIH (CA 096832). E.D. Marsh was supported by grants from the NINDS/NIH (R01 NS082761-01 and 1-KO2-NS065975).