Mark Vitucci
University of North Carolina at Chapel Hill
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Neuro-oncology | 2013
Mark Vitucci; Natalie O. Karpinich; Ryan E. Bash; Andrea M. Werneke; Ralf S. Schmid; Kristen K. White; Robert S. McNeill; Byron Huff; Sophie Wang; Terry Van Dyke; C. Ryan Miller
BACKGROUND 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. METHODS Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. RESULTS Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. CONCLUSIONS These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies.
Brain Research Bulletin | 2012
Ralf S. Schmid; Mark Vitucci; C. Ryan Miller
Over the last decade, genetically engineered mouse models have been extensively used to dissect the genetic requirements for neoplastic initiation and progression of diffuse gliomas. While these models faithfully recapitulate the histopathological features of human gliomas, comparative genomic analyses are increasingly being utilized to comprehensively assess their fidelity to recently identified molecular subtypes of these tumors. Future progress with these models will rely on incorporating insights not only from oncogenomics studies of cancer, but also from the developmental neuroscience and stem cell biology fields to design accurate and experimentally tractable models for use in translational cancer research, particularly for experimental therapeutics studies of molecularly defined subtypes of gliomas.
Neuro-oncology | 2015
Robert S. McNeill; Mark Vitucci; Jing Wu; C. Ryan Miller
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.
Neuro-oncology | 2016
Ralf S. Schmid; Jeremy M. Simon; Mark Vitucci; Robert S. McNeill; Ryan E. Bash; Andrea M. Werneke; Lauren Huey; Kristen K. White; Matthew G. Ewend; Jing Wu; C. Ryan Miller
BACKGROUND 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. METHODS To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.
Journal of Visualized Experiments | 2014
Robert S. McNeill; Ralf S. Schmid; Ryan E. Bash; Mark Vitucci; Kristen K. White; Andrea M. Werneke; Brian H. Constance; Byron Huff; C. Ryan Miller
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.
Nature Communications | 2018
Carla Danussi; Promita Bose; Prasanna Parthasarathy; Pedro Silberman; John S. Van Arnam; Mark Vitucci; Oliver Tang; Adriana Heguy; Yuxiang Wang; Timothy A. Chan; Gregory J. Riggins; Erik P. Sulman; Frederick F. Lang; Chad J. Creighton; Benjamin Deneen; C. Ryan Miller; David J. Picketts; Kasthuri Kannan; Jason T. Huse
Mutational inactivation of the SWI/SNF chromatin regulator ATRX occurs frequently in gliomas, the most common primary brain tumors. Whether and how ATRX deficiency promotes oncogenesis by epigenomic dysregulation remains unclear, despite its recent implication in both genomic instability and telomere dysfunction. Here we report that Atrx loss recapitulates characteristic disease phenotypes and molecular features in putative glioma cells of origin, inducing cellular motility although also shifting differentiation state and potential toward an astrocytic rather than neuronal histiogenic profile. Moreover, Atrx deficiency drives widespread shifts in chromatin accessibility, histone composition, and transcription in a distribution almost entirely restricted to genomic sites normally bound by the protein. Finally, direct gene targets of Atrx that mediate specific Atrx-deficient phenotypes in vitro exhibit similarly selective misexpression in ATRX-mutant human gliomas. These findings demonstrate that ATRX deficiency and its epigenomic sequelae are sufficient to induce disease-defining oncogenic phenotypes in appropriate cellular and molecular contexts.ATRX inactivation frequently occurs in glioma. Here, the authors explore the role of ATRX inactivation in oncogenesis, highlighting ATRX deficiency driven epigenomic changes that influence the expression of genes crucial to the oncogenic phenotype.
Neuro-oncology | 2017
Mark Vitucci; David M. Irvin; Robert S. McNeill; Ralf S. Schmid; Jeremy M. Simon; Harshil Dhruv; Marni B. Siegel; Andrea M. Werneke; Ryan E. Bash; Seungchan Kim; Michael E. Berens; C. R. Miller
Background Gliomas are diverse neoplasms with multiple molecular subtypes. How tumor-initiating mutations relate to molecular subtypes as these tumors evolve during malignant progression remains unclear. Methods We used genetically engineered mouse models, histopathology, genetic lineage tracing, expression profiling, and copy number analyses to examine how genomic tumor diversity evolves during the course of malignant progression from low- to high-grade disease. Results Knockout of all 3 retinoblastoma (Rb) family proteins was required to initiate low-grade tumors in adult mouse astrocytes. Mutations activating mitogen-activated protein kinase signaling, specifically KrasG12D, potentiated Rb-mediated tumorigenesis. Low-grade tumors showed mutant Kras-specific transcriptome profiles but lacked copy number mutations. These tumors stochastically progressed to high-grade, in part through acquisition of copy number mutations. High-grade tumor transcriptomes were heterogeneous and consisted of 3 subtypes that mimicked human mesenchymal, proneural, and neural glioblastomas. Subtypes were confirmed in validation sets of high-grade mouse tumors initiated by different driver mutations as well as human patient-derived xenograft models and glioblastoma tumors. Conclusion These results suggest that oncogenic driver mutations influence the genomic profiles of low-grade tumors and that these, as well as progression-acquired mutations, contribute strongly to the genomic heterogeneity across high-grade tumors.
Cancer Research | 2012
Mark Vitucci; Byron Huff; Ryan E. Bash; Natalie O. Karpinich; Ralf S. Schmid; C. Ryan Miller
Astrocytomas are characterized by diffuse invasion, precluding their complete surgical resection. PTEN, a negative PI3 kinase (PI3K) pathway regulator, is altered in 40-80% of high-grade astrocytomas (HGA), including glioblastoma (GBM). However, its role in astrocytoma invasion remains unclear. Primary astrocytes from six genetically-engineered mouse (GEM) models, with conditional alleles that inactivate Rb (T) and/or Pten (P) and/or constitutively activate Kras (R, KrasG12D) upon Cre recombination, were used to analyze PI3K pathway signaling, proliferation, migration, and invasion in vitro by immunoblot, cell counting, wound healing and time-lapse video microscopy, and collagen invasion, respectively. Gene expression microarrays were used to compare the transcriptomes of GEM astrocytes to human HGA. Tumorigenicity and survival were determined in vivo in orthotopic allograft models. Invasion was assessed by morphometric analysis. Pten ablation increased levels of phospho-Akt and phospho-S6. In cells with both Rb inactivation and Kras activation (TR), complete inactivation of Pten shortened doubling time (DT) by 42% (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4305. doi:1538-7445.AM2012-4305
Cancer Research | 2018
Carla Danussi; Promita Bose; Pedro Silberman; John S. Van Arnam; Mark Vitucci; Oliver Tang; Adriana Heguy; Timothy A. Chan; Erik P. Sulman; Frederick F. Lang; Chad J. Creighton; Benjamin Deneen; C. Ryan Miller; David J. Picketts; Kasthuri Kannan; Jason T. Huse
Neuro-oncology | 2017
Carla Danussi; Promita Bose; Prasanna Parthasarathy; Pedro Silberman; John S. Van Arnam; Mark Vitucci; Oliver Tang; Adriana Heguy; Timothy A. Chan; Erik P. Sulman; Fred Lang; Chad J. Creighton; Benjamin Deneen; C. Ryan Miller; David J. Picketts; Kasthuri Kannan; Jason T. Huse