MOLECULAR CHARACTERIZATION OF MURINE MODELS OF ASTROCYTOMA

Astrocytomas are some of the most lethal diffuse gliomas, and glioblastoma (GBM, Grade IV astrocytoma) has a median survival of 12-15 months with therapy. The last decade has seen increased efforts to define the molecular landscape of human GBM, and led to a focus on genetic abnormalities within the receptor tyrosine kinase (RTK), RB cell cycle, and P53 signaling pathways. Genetically-engineered mouse (GEM) models have been designed based upon the data from these studies and have helped determine some of the requirements for gliomagenesis depending on the cellular and developmental context. Despite these efforts gliomagenesis requirements and progression are not completely defined, and more importantly, it is often unclear which molecular subtype is modeled by these GEM. In this work, we employ GEM with conditional, inducible mutations in the RB cell cycle, MAPK, and PI3K pathways to effect astrocytoma initiation followed by stochastic progression in astrocytes throughout the brain in adult mice. We define the requirements for astrocytoma initiation and the effect they have on gene expression and copy number. Stochastic progression to high-grade astrocytoma (HGA) and GBM are characterized by detection via contrast-enhancing MRI, rapid growth, genotype-dependent survival, acquisition of copy number abnormalities (CNA), and gene expression subtypes that resemble human GBM. These subtypes correlate with brain region rather than original genotype. In parallel, we isolated astrocytes from pups containing the same genetic mutations and induced recombination in culture to create G1/S-defective astrocytes with activated Kras and/or Pten deletion. We examined how these individual and combined mutations affected gene expression and phenotypic hallmarks of astrocytoma tumorigenesis including cell growth, migration, and invasion. Combined disruption of MAPK and PI3K signaling led to the most aggressive, invasive astrocytes (TRP) with stem-like and proneural expression profiles. These TRP astrocytes were confirmed to have stem cell properties in vitro and in vivo. After orthotopic injection into syngeneic mice, these TRP astrocytes formed HGA with high incidence, short latency, and reproducible survival, supporting its utility as a preclinical model. We replicated standard of care GBM treatment consisting of radiation with concurrent temozolomide and showed that TRP allografts were susceptible to radiation but not temozolomide. Similar to TRP astrocytes in vitro, the allograft HGA expression profiles were proneural, but after radiation treatment most were most similar to the mesenchymal subtype. Overall, this research defines the requirements for astrocytoma in adult murine astrocytes and raises important questions about whether mutations, cell type, or location determines molecular subtype. We develop several models which will be useful to further elucidate the molecular nuances of astrocytoma and their effects on initiation, progression, and signaling pathways. These models will also serve as the basis for future subtype specific preclinical models in which to develop novel gene signatures, biomarkers, and molecularly targeted therapies.Doctor of Philosoph

Contemporary murine models in preclinical astrocytoma drug development

Despite 6 decades of research, only 3 drugs have been approved for astrocytomas, the most common malignant primary brain tumors. However, clinical drug development is accelerating with the transition from empirical, cytotoxic therapy to precision, targeted medicine. Preclinical animal model studies are critical for prioritizing drug candidates for clinical development and, ultimately, for their regulatory approval. For decades, only murine models with established tumor cell lines were available for such studies. However, these poorly represent the genomic and biological properties of human astrocytomas, and their preclinical use fails to accurately predict efficacy in clinical trials. Newer models developed over the last 2 decades, including patient-derived xenografts, genetically engineered mice, and genetically engineered cells purified from human brains, more faithfully phenocopy the genomics and biology of human astrocytomas. Harnessing the unique benefits of these models will be required to identify drug targets, define combination therapies that circumvent inherent and acquired resistance mechanisms, and develop molecular biomarkers predictive of drug response and resistance. With increasing recognition of the molecular heterogeneity of astrocytomas, employing multiple, contemporary models in preclinical drug studies promises to increase the efficiency of drug development for specific, molecularly defined subsets of tumors

Core pathway mutations induce de-differentiation of murine astrocytes into glioblastoma stem cells that are sensitive to radiation but resistant to temozolomide

Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype

Modeling Astrocytoma Pathogenesis <em>In Vitro</em> and <em>In Vivo</em> Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases

Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies

De novo single-nucleotide and copy number variation in discordant monozygotic twins reveals disease-related genes

Recent studies have demonstrated genetic differences between monozygotic (MZ) twins. To test the hypothesis that early post-twinning mutational events associate with phenotypic discordance, we investigated a cohort of 13 twin pairs (n = 26) discordant for various clinical phenotypes using whole-exome sequencing and screened for copy number variation (CNV). We identified a de novo variant in PLCB1, a gene involved in the hydrolysis of lipid phosphorus in milk from dairy cows, associated with lactase non-persistence, and a variant in the mitochondrial complex I gene MT-ND5 associated with amyotrophic lateral sclerosis (ALS). We also found somatic variants in multiple genes (TMEM225B, KBTBD3, TUBGCP4, TFIP11) in another MZ twin pair discordant for ALS. Based on the assumption that discordance between twins could be explained by a common variant with variable penetrance or expressivity, we screened the twin samples for known pathogenic variants that are shared and identified a rare deletion overlapping ARHGAP11B, in the twin pair manifesting with either schizotypal personality disorder or schizophrenia. Parent-offspring trio analysis was implemented for two twin pairs to assess potential association of variants of parental origin with susceptibility to disease. We identified a de novo variant in RASD2 shared by 8-year-old male twins with a suspected diagnosis of autism spectrum disorder (ASD) manifesting as different traits. A de novo CNV duplication was also identified in these twins overlapping CD38, a gene previously implicated in ASD. In twins discordant for Tourette's syndrome, a paternally inherited stop loss variant was detected in AADAC, a known candidate gene for the disorder

Supporting data

Excel files containing data used in publicatio

Data from: Primary care clinics can be a source of exposure to virulent Clostridium (now Clostridioides) difficile: an environmental screening study of hospitals and clinics in Dallas-Fort Worth region

C. difficile is an endospore-forming pathogen, which is becoming a common cause of microbial health-care associated gastrointestinal disease in the United States. Both healthy and symptomatic patients can shed C. difficile spores into the environment, which can survive for long periods, being resistant to desiccation, heat, and disinfectants. In healthcare facilities, environmental contamination with C. difficile is a major concern as a potential source of exposure to this pathogen and risk of disease in susceptible patients. Although hospital-acquired infection is recognized, community-acquired infection is an increasingly recognized health problem. Primary care clinics may be a significant source of exposure to this pathogen; however, there are limited data about presence of environmental C. difficile within clinics. To address the potential for primary care clinics as a source of environmental exposure to virulent C. difficile, we measured the frequency of environmental contamination with spores in clinic examination rooms and hospital rooms in Dallas-Fort Worth (DFW) area of Texas. The ribotypes and presence of toxin genes from some environmental isolates were compared. Our results indicate primary care clinics have higher frequencies of contamination than hospitals. After notification of the presence of C. difficile spores in the clinics and an educational discussion to emphasize the importance of this infection and methods of infection prevention, environmental contamination in clinics was reduced on subsequent sampling to that found in hospitals. Thus, primary care clinics can be a source of exposure to virulent C. difficile, and recognition of this possibility can result in improved infection prevention, potentially reducing community-acquired C. difficile infections and subsequent disease

Primary care clinics can be a source of exposure to virulent Clostridium (now Clostridioides) difficile: An environmental screening study of hospitals and clinics in Dallas-Fort Worth region.

C. difficile is an endospore-forming pathogen, which is becoming a common cause of microbial health-care associated gastrointestinal disease in the United States. Both healthy and symptomatic patients can shed C. difficile spores into the environment, which can survive for long periods, being resistant to desiccation, heat, and disinfectants. In healthcare facilities, environmental contamination with C. difficile is a major concern as a potential source of exposure to this pathogen and risk of disease in susceptible patients. Although hospital-acquired infection is recognized, community-acquired infection is an increasingly recognized health problem. Primary care clinics may be a significant source of exposure to this pathogen; however, there are limited data about presence of environmental C. difficile within clinics. To address the potential for primary care clinics as a source of environmental exposure to virulent C. difficile, we measured the frequency of environmental contamination with spores in clinic examination rooms and hospital rooms in Dallas-Fort Worth (DFW) area of Texas. The ribotypes and presence of toxin genes from some environmental isolates were compared. Our results indicate primary care clinics have higher frequencies of contamination than hospitals. After notification of the presence of C. difficile spores in the clinics and an educational discussion to emphasize the importance of this infection and methods of infection prevention, environmental contamination in clinics was reduced on subsequent sampling to that found in hospitals. Thus, primary care clinics can be a source of exposure to virulent C. difficile, and recognition of this possibility can result in improved infection prevention, potentially reducing community-acquired C. difficile infections and subsequent disease