Karlyne M. Reilly

Nf1 ; Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects

Astrocytomas are the leading cause of brain cancer in humans. Because these tumours are highly infiltrative, current treatments that rely on targeting the tumour mass are often ineffective. A mouse model for astrocytoma would be a powerful tool for dissecting tumour progression and testing therapeutics. Mouse models of astrocytoma have been designed to express oncogenic proteins in astrocytes, but have had limited success due to low tumour penetrance or limited tumour progression. We present here a mouse model of astrocytomas involving mutation of two tumour-suppressor genes, Nf1 and Trp53. Humans with mutations in NF1 develop neurofibromatosis type I (NF1) and have increased risk of optic gliomas, astrocytomas and glioblastomas. The TP53 tumour suppressor is often mutated in a subset of astrocytomas that develop at a young age and progress slowly to glioblastoma (termed secondary glioblastomas, in contrast to primary glioblastomas that develop rapidly de novo). This mouse model shows a range of astrocytoma stages, from low-grade astrocytoma to glioblastoma multiforme, and may accurately model human secondary glioblastoma involving TP53 loss. This is the first reported mouse model of astrocytoma initiated by loss of tumour suppressors, rather than overexpression of transgenic oncogenes.

Neurofibromatosis 1 (NF1) heterozygosity results in a cell‐autonomous growth advantage for astrocytes

Individuals with neurofibromatosis 1 (NF1) develop low‐grade astrocytomas at an increased frequency. To gain insight into the function of the Nf1 gene product as a growth regulator for astrocytes, we examined mice heterozygous for a targeted Nf1 mutation. In our previous studies, we demonstrated increased numbers of proliferating astrocytes in Nf1 heterozygote (Nf1+/−) mice in vivo. We now show that cultured Nf1+/− astrocytes exhibit a cell‐autonomous growth advantage in vitro associated with increased p21‐ras pathway activation. Furthermore, we demonstrate that Nf1+/−;wild‐type N‐ras mice have a similar astrocyte growth advantage in vitro and in vivo as either oncogenic N‐ras or Nf1+/−;oncogenic N‐ras mice. Lastly, mice heterozygous for targeted defects in both Nf1 and p53 as well as Nf1 and Rb exhibit 3‐ and 2.5‐fold increases in astrocyte proliferation in vivo, respectively, suggesting that abnormalities in Nf1‐ and p53/Rb‐regulated pathways cooperate in the heterozygous state to confer a growth advantage for brain astrocytes. Collectively, these results provide evidence for a cell‐autonomous growth advantage in Nf1+/− astrocytes and suggest that some of the brain pathology in individuals with NF1 might result from reduced, but not absent, NF1 gene function. GLIA 33:314–323, 2001.

Comparative Pathology of Nerve Sheath Tumors in Mouse Models and Humans

Despite the progress made in our understanding of the biology of neurofibromatosis (NF), the long-term clinical outcome for affected patients has not changed significantly in the past decades, and both NF1 and NF2 are still associated with a significant morbidity and a decreased life span. A number of NF1 and NF2 murine models have been generated to aid in the study of NF tumor biology and in the development of targeted therapies for NF patients. A single, universal pathological classification of the lesions generated in these murine models is essential for the validation of the models, for their analysis and comparison with other models, and for their future effective use in preclinical treatment trials. For the formulation of a pathological classification of these lesions, the WHO classification of human tumors was used as a reference. However, it was not adopted for the classification of the GEM lesions because of some important differences between the human and murine lesions. A novel classification scheme for peripheral nerve sheath tumors in murine models was therefore devised.

Genetically engineered mouse models in cancer research.

Mouse models of human cancer have played a vital role in understanding tumorigenesis and answering experimental questions that other systems cannot address. Advances continue to be made that allow better understanding of the mechanisms of tumor development, and therefore the identification of better therapeutic and diagnostic strategies. We review major advances that have been made in modeling cancer in the mouse and specific areas of research that have been explored with mouse models. For example, although there are differences between mice and humans, new models are able to more accurately model sporadic human cancers by specifically controlling timing and location of mutations, even within single cells. As hypotheses are developed in human and cell culture systems, engineered mice provide the most tractable and accurate test of their validity in vivo. For example, largely through the use of these models, the microenvironment has been established to play a critical role in tumorigenesis, since tumor development and the interaction with surrounding stroma can be studied as both evolve. These mouse models have specifically fueled our understanding of cancer initiation, immune system roles, tumor angiogenesis, invasion, and metastasis, and the relevance of molecular diversity observed among human cancers. Currently, these models are being designed to facilitate in vivo imaging to track both primary and metastatic tumor development from much earlier stages than previously possible. Finally, the approaches developed in this field to achieve basic understanding are emerging as effective tools to guide much needed development of treatment strategies, diagnostic strategies, and patient stratification strategies in clinical research.

Neuropathology of genetically engineered mice: consensus report and recommendations from an international forum

The Mouse Models of Cancer Consortium of the NCI sponsored a meeting of neuropathologists and veterinary pathologists in New York City in November of 2000. A rapidly growing number of genetically engineered mice (GEM) predisposed to tumors of the nervous system have led to a concomitant need for neuropathological evaluation and validation of these models. A panel of 13 pathologists reviewed material representing most of the available published and unpublished GEM models of medulloblastoma, primitive neuroectodermal tumor, astrocytoma, oligodendroglioma, mixed glioma, and tumors of the peripheral nerve. The GEM tumors were found to have many similarities and some distinct differences with respect to human disease. After review of the biology and pathology for all models presented, participants were split into groups reflective of clinical expertise in human pathology, tumor biology, neuroimaging, or treatment/intervention. Recommendations were made detailing an extensive and complete neuropathological characterization of animals. Importance was placed on including information on strains, tumor clonality, and examination for genetic mutation or altered gene expression characteristics of the corresponding human malignancy. Specific proposals were made to incorporate GEM models in emerging neuroradiological modalities. Recommendations were also made for preclinical validation of these models in cancer therapeutics, and for incorporation of surrogate markers of tumor burden to facilitate preclinical evaluation of new therapies.

The neurofibromatosis type 1 tumor suppressor controls cell growth by regulating signal transducer and activator of transcription-3 activity in vitro and in vivo

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome in which affected individuals develop benign and malignant nerve tumors. The NF1 gene product neurofibromin negatively regulates Ras and mammalian target of rapamycin (mTOR) signaling, prompting clinical trials to evaluate the ability of Ras and mTOR pathway inhibitors to arrest NF1-associated tumor growth. To discover other downstream targets of neurofibromin, we performed an unbiased cell-based high-throughput chemical library screen using NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells. We identified the natural product, cucurbitacin-I (JSI-124), which inhibited NF1-deficient cell growth by inducing apoptosis. We further showed that signal transducer and activator of transcription-3 (STAT3), the target of cucurbitacin-I inhibition, was hyperactivated in NF1-deficient primary astrocytes and neural stem cells, mouse glioma cells, and human MPNST cells through Ser(727) phosphorylation, leading to increased cyclin D1 expression. STAT3 was regulated in NF1-deficient cells of murine and human origin in a TORC1- and Rac1-dependent manner. Finally, cucurbitacin-I inhibited the growth of NF1-deficient MPNST cells in vivo. In summary, we used a chemical genetics approach to reveal STAT3 as a novel neurofibromin/mTOR pathway signaling molecule, define its action and regulation, and establish STAT3 as a tractable target for future NF1-associated cancer therapy studies.

An Imprinted Locus Epistatically Influences Nstr1 and Nstr2 to Control Resistance to Nerve Sheath Tumors in a Neurofibromatosis Type 1 Mouse Model

Cancer is a complex disease in which cells acquire many genetic and epigenetic alterations. We have examined how three types of alterations, mutations in tumor suppressor genes, changes in an imprinted locus, and polymorphic loci, interact to affect tumor susceptibility in a mouse model of neurofibromatosis type 1 (NF1). Mutations in tumor suppressor genes such as TP53 and in oncogenes such as KRAS have major effects on tumorigenesis due to the central roles of these genes in cell proliferation and cell survival. Imprinted genes expressed from only one parental chromosome affect tumorigenesis if their monoallelic expression is lost or duplicated. Because imprinted loci are within regions deleted or amplified in cancer, the parental origin of genomic rearrangements could affect tumorigenesis. Gene polymorphisms can vary tumor incidence by affecting rate-limiting steps in tumorigenesis within tumor cells or surrounding stroma. In our mouse model of NF1, the incidence of tumors mutant for the tumor suppressor genes Nf1 and Trp53 is strongly modified by a linked imprinted locus acting epistatically on two unlinked polymorphic loci, Nstr1 and Nstr2. This interaction of an imprinted locus and polymorphic susceptibility loci has profound implications for human mapping studies where the parental contribution of alleles is often unknown.

Nf1 expression is dependent on strain background: implications for tumor suppressor haploinsufficiency studies

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.

Schweinfurthin A Selectively Inhibits Proliferation and Rho Signaling in Glioma and Neurofibromatosis Type 1 Tumor Cells in a NF1-GRD–Dependent Manner

Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1−/+;Trp53−/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1−/−;Trp53−/− astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor–stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling. Mol Cancer Ther; 9(5); 1234–43. ©2010 AACR.

Chr 19A/J modifies tumor resistance in a sex and parent-of-origin specific manner

Neurofibromatosis type 1 (NF1) is one of the most common human genetic diseases affecting the nervous system and predisposes individuals to cancer, including peripheral nerve sheath tumors (PNSTs) and astrocytomas. Modifiers in the genetic background affect the severity of the disease and we have previously mapped two modifier loci, Nstr1 and Nstr2, that influence resistance to PNSTs in the Nf1−/+;Trp53−/+cis mouse model of NF1. We report here the analysis of Nstr1 in isolation from other epistatic loci using a chromosome substitution strain, and further show that a modifier locus (or loci) on chromosome 19 influences resistance to both PNSTs and astrocytomas. This modifier locus interacts with sex, resulting in sex-specific modification of tumors. Allele variability on chromosome 19 affects both the timing and the penetrance of the growth of different tumor types associated with NF1, specifically PNSTs and astrocytoma. These results indicate that modifiers of cancer susceptibility interact and affect tumorigenesis under different genetic conditions and demonstrate the power of chromosome substitution strains to study genetic modifiers.