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BRAF Mutations Open Doors for N-Ethyl-N-Nitrosourea–Induced Gliomagenesis

  • Robert S. McNeill
    Affiliations
    Pathobiology and Translational Science Graduate Program, University of North Carolina School of Medicine, Chapel Hill, North Carolina
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  • David M. Irvin
    Affiliations
    Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina
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  • C. Ryan Miller
    Correspondence
    Address correspondence to C. Ryan Miller, M.D., Ph.D., University of North Carolina School of Medicine, 6109B Neurosciences Research Bldg, Campus Box 7250, Chapel Hill, NC 27599-7250.
    Affiliations
    Pathobiology and Translational Science Graduate Program, University of North Carolina School of Medicine, Chapel Hill, North Carolina

    Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina

    Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina

    Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina

    Department of Neurology and Neurosciences Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina
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Open ArchivePublished:August 18, 2016DOI:https://doi.org/10.1016/j.ajpath.2016.07.005
      In this issue of the American Journal of Pathology, Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      identified recurrent Braf mutations in N-ethyl-N-nitrosourea (ENU)–induced rat gliomas by DNA sequencing. Their results provide a platform for preclinical development of novel targeted therapies for BRAF-mutant gliomas. Precision oncology promises to revolutionize cancer therapy by stratifying tumors on the basis of their molecular characteristics and using rationally designed treatments in molecularly defined patient populations.
      • Mendelsohn J.
      Personalizing oncology: perspectives and prospects.
      Although the field of oncology is transitioning into the precision medicine era, conventional diagnostics, based on tissue type, tumor pathology, and patient demographics, remain essential to quality care. One natural pathological division for gliomas, the most common primary brain tumors in humans, is based on their invasion, with nondiffuse gliomas forming well-circumscribed tumors and diffuse gliomas invading the normal brain.
      • Louis D.N.
      • Perry A.
      • Reifenberger G.
      • von Deimling A.
      • Figarella-Branger D.
      • Cavenee W.K.
      • Ohgaki H.
      • Wiestler O.D.
      • Kleihues P.
      • Ellison D.W.
      The 2016 World Health Organization classification of tumors of the central nervous system: a summary.
      Within these two broad diagnostic categories, gliomas are further subdivided on the basis of patient demographics into pediatric and adult diseases and further into specific diagnostic entities based on their histological appearance.

      Human Gliomas Are Genomically Heterogeneous

      Genomic analyses are necessary to clinically implement the concepts of precision medicine. Comprehensive genomic studies have revealed the genetic diversity of specific glioma entities and increased precision in defining relevant disease subtypes. For instance, integration of multiple genomic and molecular analyses determined that there are three molecular classes of adult lower-grade gliomas (grade II and III astrocytomas, oligoastrocytomas, and oligodendrogliomas, based on the World Health Organization 2007 classification scheme) that are more accurately represented by genetic testing for IDH1/2, ATRX, and TP53 mutations and chromosome 1p and 19q losses than by histological class.
      • Louis D.N.
      • Perry A.
      • Reifenberger G.
      • von Deimling A.
      • Figarella-Branger D.
      • Cavenee W.K.
      • Ohgaki H.
      • Wiestler O.D.
      • Kleihues P.
      • Ellison D.W.
      The 2016 World Health Organization classification of tumors of the central nervous system: a summary.
      • Cancer Genome Atlas Research Network
      Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas.
      Large-scale genomic sequencing has also shown that pediatric gliomas are genomically distinct diseases compared to their histologically similar adult counterparts.
      • Paugh B.S.
      • Qu C.
      • Jones C.
      • Liu Z.
      • Adamowicz-Brice M.
      • Zhang J.
      • Bax D.A.
      • Coyle B.
      • Barrow J.
      • Hargrave D.
      • Lowe J.
      • Gajjar A.
      • Zhao W.
      • Broniscer A.
      • Ellison D.W.
      • Grundy R.G.
      • Baker S.J.
      Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease.
      Moreover, genomic analyses have shown that even within a single demographic group or histological entity, multiple molecular subtypes of disease exist.
      • Brennan C.W.
      • Verhaak R.G.
      • McKenna A.
      • Campos B.
      • Noushmehr H.
      • Salama S.R.
      • et al.
      The somatic genomic landscape of glioblastoma.
      • Saratsis A.M.
      • Kambhampati M.
      • Snyder K.
      • Yadavilli S.
      • Devaney J.M.
      • Harmon B.
      • Hall J.
      • Raabe E.H.
      • An P.
      • Weingart M.
      • Rood B.R.
      • Magge S.N.
      • MacDonald T.J.
      • Packer R.J.
      • Nazarian J.
      Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes.
      Our increased understanding of the molecular characteristics underpinning glioma pathogenesis and increased precision in defining disease subsets will hopefully lead to the development of treatments that target the molecular aberrations driving their genesis. However, to fully achieve the promise of precision medicine, it will be necessary to use preclinical models that match specific disease subsets not only in terms of pathology and demographics, but on genetic mechanisms as well.

      Modeling Gliomas Using ENU

      The first animal models of human gliomas were developed by treating either mice or rats with chemical carcinogens.
      • McNeill R.S.
      • Vitucci M.
      • Wu J.
      • Miller C.R.
      Contemporary murine models in preclinical astrocytoma drug development.
      One such carcinogen is ENU, a DNA ethylating agent that induces widespread DNA damage, resulting in single-nucleotide mutations, most commonly T:A to A:T transversions and T:A to C:G transitions.
      • Justice M.J.
      • Noveroske J.K.
      • Weber J.S.
      • Zheng B.
      • Bradley A.
      Mouse enu mutagenesis.
      Rats in particular have been treated with ENU either during embryonic development or postnatally to induce gliomagenesis, and the resulting gliomas were either characterized within the intact brain or harvested to establish adherent cell lines cultured in vitro in serum-containing media.
      Serum-cultured cell lines established from either chemically induced rodent gliomas or naturally occurring human tumors (established cell lines) have been the backbone of preclinical glioma research since the late 1960s.
      • McNeill R.S.
      • Vitucci M.
      • Wu J.
      • Miller C.R.
      Contemporary murine models in preclinical astrocytoma drug development.
      These models were widely disseminated and routinely used, but their genetic make-up was largely unknown until the advent of large-scale genomic analyses, such as microarray profiling and next-generation sequencing. These techniques have shown that established cell lines harbor more extensive genomic abnormalities than human gliomas and do not faithfully recapitulate the molecular profiles of patient samples.
      • Clark M.J.
      • Homer N.
      • O'Connor B.D.
      • Chen Z.
      • Eskin A.
      • Lee H.
      • Merriman B.
      • Nelson S.F.
      U87mg decoded: the genomic sequence of a cytogenetically aberrant human cancer cell line.
      • Li A.
      • Walling J.
      • Kotliarov Y.
      • Center A.
      • Steed M.E.
      • Ahn S.J.
      • Rosenblum M.
      • Mikkelsen T.
      • Zenklusen J.C.
      • Fine H.A.
      Genomic changes and gene expression profiles reveal that established glioma cell lines are poorly representative of primary human gliomas.
      Reasons for these discrepancies include adaptation to nutrient-rich media and clonal selection.
      • McNeill R.S.
      • Vitucci M.
      • Wu J.
      • Miller C.R.
      Contemporary murine models in preclinical astrocytoma drug development.
      Nevertheless, gliomas that arise spontaneously within an intact rat brain after ENU treatment are not subject to the selection pressures of serum culture. Therefore, these models may more faithfully recapitulate the genomics of acutely isolated patient tumors. Whether mutations in certain genes are highly penetrant or whether multiple mutations converge on specific, common biological pathway(s) in ENU-induced gliomas remained unclear.

      ENU-Induced Rat Gliomas Harbor Mutant Braf

      The study by Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      determined the mutational profile of ENU-induced rat gliomas.
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      Whole genome sequencing was performed on tumors from three BDIV and two BDIX rats induced with ENU at post-natal day 1. Somatic mutations ranged from 6354 to 13,807 per glioma, with a mean of 10,685, but recurrent copy number alterations were absent in the tumors examined. Consistent with ENU-induced mutagenesis, the most common single-nucleotide mutations were T:A to A:T transversions and T:A to C:G transitions. Between 35 and 93 of these somatic mutations were shown to be functional, causing amino acid changes (92%), introduction of stop codons (6%), or destruction of splice sites (2%). Somatic mutations in Srrm2, Olr158, Il12rb, Map1b, Runx2, Rsrc2, and Tcf21 occurred in two of the five gliomas analyzed. The only mutation unique to all five was an A to T missense mutation corresponding to BrafV545E. Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      found that this mutation was analogous to the BRAFV600E mutation at both the nucleotide and amino acid levels in humans and mice. To confirm the occurrence of the BrafV545E in ENU-induced rat gliomas, the authors expanded their cohort to another 33 BDIV and 12 BDIX gliomas. Sanger sequencing determined that all 45 also harbored the BrafV545E mutation. Moreover, BrafV545E mutant rat gliomas were immunoreactive to a human BRAFV600E antibody. Thus, they found that BrafV545E is a ubiquitous mutation in ENU-induced gliomas in rats.

      Role of BRAF in Tumorigenesis

      BRAF encodes a serine/threonine protein kinase that activates the mitogen activated protein kinase effector arm of receptor tyrosine kinase signaling. Activating BRAF mutations, particularly the most common V600E mutation, have been implicated in tumorigenesis in a variety of solid cancers, including melanoma (approximately 70%), papillary thyroid cancer (45%), and colorectal cancers (approximately 10%). This mutation lies within the kinase domain and leads to constitutive activation of the protein and hyperactivation of mitogen activated protein kinase signaling.
      • Cantwell-Dorris E.R.
      • O'Leary J.J.
      • Sheils O.M.
      Brafv600e: implications for carcinogenesis and molecular therapy.
      Genomic analyses have shown that BRAF mutations occur in nondiffuse human gliomas, including pilocytic astrocytomas, gangliogliomas, and pleomorphic xanthoastrocytoma.
      • Horbinski C.
      To braf or not to braf: is that even a question anymore?.
      More important, all three tumor types develop most commonly in children and young adults and may display the cytological features of their diffuse oligodendroglioma counterparts, including perinuclear halos, crisp nuclear membranes, and prominent nucleoli. However, unlike diffuse oligodendrogliomas, each of these tumors are slow growing, benign (World Health Organization grade I, pilocytic astrocytomas and gangliogliomas) to mildly aggressive (World Health Organization grade II, pleomorphic xanthoastrocytoma), and have discrete tumor margins. They are thus generally surgically curable. Nevertheless, when location precludes complete resection, adjuvant therapies are required. Drugs targeting BRAFV600E (BRAFi) have been approved for treatment of metastatic melanoma.
      • Bollag G.
      • Tsai J.
      • Zhang J.
      • Zhang C.
      • Ibrahim P.
      • Nolop K.
      • Hirth P.
      Vemurafenib: the first drug approved for braf-mutant cancer.
      However, resistance inevitably occurs through a variety of mechanisms, including reactivation of mitogen activated protein kinase signaling or alternate activation of parallel pathways. Thus, rationally designed combination therapies that target multiple kinases within the same pathway and/or alternate pathways may be necessary to circumvent BRAFi resistance. Moreover, BRAFi have shown mixed results in the treatment of BRAF-mutant gliomas.
      • Collins V.P.
      • Jones D.T.
      • Giannini C.
      Pilocytic astrocytoma: pathology, molecular mechanisms and markers.
      Case reports suggest that BRAFV600E-mutant gliomas are sensitive to BRAFi, but this observation has not been confirmed in clinical trials. Developing more accurate preclinical models to determine the underlying biology of BRAF-mutant gliomas is necessary for efficient development of novel treatments.
      Historically, the histological and cytological features of ENU-induced rat gliomas, including the extent of brain invasion, have been variable, with most being diagnosed as oligodendrogliomas, astrocytomas, or mixed gliomas.
      • Zook B.C.
      • Simmens S.J.
      • Jones R.V.
      Evaluation of enu-induced gliomas in rats: nomenclature, immunochemistry, and malignancy.
      The ENU-induced rat gliomas from the study by Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      showed cytological features similar to low-grade or anaplastic oligodendrogliomas, but lacked diffuse brain invasion.
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      Taken together, the histopathology of these model tumors is consistent with the nondiffuse gliomas, such as pilocytic astrocytomas, gangliogliomas, or pleomorphic xanthoastrocytoma, and their lack of invasion further supports the use of this model for studying these entities. Indeed, a significant strength of this ENU-induced rat glioma model is the developmental timing of tumorigenesis that may mimic pediatric patients.
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      ENU injections were performed in perinatal mice, mirroring the age at which BRAFV600E gliomas might be initiated in humans. This timing, plus the ubiquitous penetrance of Braf V545E mutations, suggests that this preclinical model may prove beneficial in studying the basic biology of BRAF-mutant pediatric gliomas and developing targeted therapies.

      Using BRAF-Mutant Murine Models to Study Gliomagenesis and Develop Targeted Therapies

      The complete penetrance of BRAFV545E mutations in the ENU-induced rat gliomas described by Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      suggests that this mutation drives tumorigenesis. However, the study was underpowered to identify other significantly mutated genes that may cooperate with mutant BRAF.
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      Future studies using whole exome sequencing, sophisticated statistical tools, and increased samples sizes could confirm the frequency of the BRAFV545E mutation and identify potential cooperating mutations, such as CDKN2A (Ink4a-Arf) loss, that frequently co-occur in human gliomas.
      • Lawrence M.S.
      • Stojanov P.
      • Polak P.
      • Kryukov G.V.
      • Cibulskis K.
      • Sivachenko A.
      • et al.
      Mutational heterogeneity in cancer and the search for new cancer-associated genes.
      • Huillard E.
      • Hashizume R.
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      • Aoki Y.
      • Nicolaides T.
      • Perry A.
      • Waldman T.
      • McMahon M.
      • Weiss W.A.
      • Petritsch C.
      • James C.D.
      • Rowitch D.H.
      Cooperative interactions of brafv600e kinase and cdkn2a locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy.
      • Schindler G.
      • Capper D.
      • Meyer J.
      • Janzarik W.
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      • Korshunov A.
      • Pfister S.
      • Hartmann C.
      • Paulus W.
      • Reifenberger G.
      • von Deimling A.
      Analysis of braf v600e mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma.
      In this regard, the role of BRAF mutations in gliomagenesis has been explored experimentally. Overexpression of both wild-type BRAF and BRAFV600E in immortalized astrocytes induces senescence in vitro.
      • Jacob K.
      • Quang-Khuong D.A.
      • Jones D.T.
      • Witt H.
      • Lambert S.
      • Albrecht S.
      • Witt O.
      • Vezina C.
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      • Hawkins C.
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      • Collins V.P.
      • Pfister S.M.
      • Tabori U.
      • Jabado N.
      Genetic aberrations leading to mapk pathway activation mediate oncogene-induced senescence in sporadic pilocytic astrocytomas.
      One study using genetically engineered mice found that BRAFV600E alone was not sufficient to induce gliomas, but cooperated with Cdkn2a deletion to do so.
      • Robinson J.P.
      • VanBrocklin M.W.
      • Guilbeault A.R.
      • Signorelli D.L.
      • Brandner S.
      • Holmen S.L.
      Activated braf induces gliomas in mice when combined with ink4a/arf loss or akt activation.
      Moreover, gliomas from these genetically engineered mice were well demarcated and noninfiltrative. In addition, the BRAFV600E kinase domain alone was sufficient to generate pilocytic astrocytoma when expressed in situ.
      • Gronych J.
      • Korshunov A.
      • Bageritz J.
      • Milde T.
      • Jugold M.
      • Hambardzumyan D.
      • Remke M.
      • Hartmann C.
      • Witt H.
      • Jones D.T.
      • Witt O.
      • Heiland S.
      • Bendszus M.
      • Holland E.C.
      • Pfister S.
      • Lichter P.
      An activated mutant braf kinase domain is sufficient to induce pilocytic astrocytoma in mice.
      Both of these studies examined the role of BRAFV600E in transformation of neonatal mouse neural stem cells. However, the role of BRAFV600E in gliomagenesis could differ on the basis of the cell of origin. The cellular origins of Braf-mutant, ENU-induced rat gliomas described in Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      remain unknown. Future studies should focus on elucidating the influence of cellular origin and cooperating mutations in BRAF-driven gliomagenesis.
      Genetically faithful, BRAF-mutant preclinical glioma models will aid in the development of novel, targeted treatments. Preclinical data suggest that BRAF is a viable target in BRAF-mutant gliomas. Indeed, genetic ablation of BRAF with shRNA reduces growth of BRAFV600E-mutant glioma cell lines.
      • Nicolaides T.P.
      • Li H.
      • Solomon D.A.
      • Hariono S.
      • Hashizume R.
      • Barkovich K.
      • Baker S.J.
      • Paugh B.S.
      • Jones C.
      • Forshew T.
      • Hindley G.F.
      • Hodgson J.G.
      • Kim J.S.
      • Rowitch D.H.
      • Weiss W.A.
      • Waldman T.A.
      • James C.D.
      Targeted therapy for brafv600e malignant astrocytoma.
      This suggests that BRAF mutations are involved in tumor maintenance as well as initiation. The role of BRAFV600E in tumor maintenance has also been investigated pharmacologically. BRAFV600E-mutant, patient-derived xenografts and established cell lines are sensitive to the BRAFi PLX4720 in vitro.
      • Nicolaides T.P.
      • Li H.
      • Solomon D.A.
      • Hariono S.
      • Hashizume R.
      • Barkovich K.
      • Baker S.J.
      • Paugh B.S.
      • Jones C.
      • Forshew T.
      • Hindley G.F.
      • Hodgson J.G.
      • Kim J.S.
      • Rowitch D.H.
      • Weiss W.A.
      • Waldman T.A.
      • James C.D.
      Targeted therapy for brafv600e malignant astrocytoma.
      • Dasgupta T.
      • Olow A.K.
      • Yang X.
      • Hashizume R.
      • Nicolaides T.P.
      • Tom M.
      • Aoki Y.
      • Berger M.S.
      • Weiss W.A.
      • Stalpers L.J.
      • Prados M.
      • James C.D.
      • Mueller S.
      • Haas-Kogan D.A.
      Survival advantage combining a braf inhibitor and radiation in braf v600e-mutant glioma.
      Moreover, intracranial genetically engineered mouse–derived allografts developed using BrafV600E driven, Ink4a-Arf null neural progenitor cells were also sensitive to PLX4720.
      • Huillard E.
      • Hashizume R.
      • Phillips J.J.
      • Griveau A.
      • Ihrie R.A.
      • Aoki Y.
      • Nicolaides T.
      • Perry A.
      • Waldman T.
      • McMahon M.
      • Weiss W.A.
      • Petritsch C.
      • James C.D.
      • Rowitch D.H.
      Cooperative interactions of brafv600e kinase and cdkn2a locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy.
      These results implicate oncogene addiction to BRAFV600E in gliomas, suggesting that the rat gliomas described by Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      may be addicted to BRAF mutations as well.
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      Despite the promise of single agent–targeted therapies, drug resistance has limited their therapeutic benefits in melanoma patients. Given the resistance patterns seen clinically, it is unlikely that BRAF-targeted therapies will be effective as single agents in gliomas.
      • Bollag G.
      • Tsai J.
      • Zhang J.
      • Zhang C.
      • Ibrahim P.
      • Nolop K.
      • Hirth P.
      Vemurafenib: the first drug approved for braf-mutant cancer.
      Therefore, the generation of rationally designed combination treatments is paramount. To this end, the use of genetically accurate preclinical model systems is necessary. Indeed, the BRAFi, PLX4720, has been tested in vivo in combination with radiation in BRAFV600E mutant patient-derived xenografts and in combination with the cyclin-dependent kinase 4/6 inhibitor PD0332991 in BRAFV600E mutant, Ink4a/Arf deleted genetically engineered mouse–derived allografts.
      • Huillard E.
      • Hashizume R.
      • Phillips J.J.
      • Griveau A.
      • Ihrie R.A.
      • Aoki Y.
      • Nicolaides T.
      • Perry A.
      • Waldman T.
      • McMahon M.
      • Weiss W.A.
      • Petritsch C.
      • James C.D.
      • Rowitch D.H.
      Cooperative interactions of brafv600e kinase and cdkn2a locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy.
      • Dasgupta T.
      • Olow A.K.
      • Yang X.
      • Hashizume R.
      • Nicolaides T.P.
      • Tom M.
      • Aoki Y.
      • Berger M.S.
      • Weiss W.A.
      • Stalpers L.J.
      • Prados M.
      • James C.D.
      • Mueller S.
      • Haas-Kogan D.A.
      Survival advantage combining a braf inhibitor and radiation in braf v600e-mutant glioma.
      These preclinical studies showed that combination treatment improved survival compared to untreated and PLX4720-treated mice. Nevertheless, all mice eventually succumbed to disease. Thus, more preclinical studies using experimentally tractable models are required to identify effective, rationally designed combination therapies. Should ENU-induced gliomas prove to have the same cooperating mutations as BRAF-mutant human gliomas, use of tumor cells harvested from this model for orthotopic implantation in a manner similar to genetically engineered mouse–derived allografts may prove useful.
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      Adapting the ENU model to culture and allografts will enable control of critical variables for preclinical drug studies, such as tumor latency and penetrance.
      • McNeill R.S.
      • Vitucci M.
      • Wu J.
      • Miller C.R.
      Contemporary murine models in preclinical astrocytoma drug development.
      Moreover, leveraging a panel of BrafV545E-mutant, ENU-induced rat gliomas harboring a variety of cooperating mutations will be useful in further elucidating BRAFi resistance mechanisms in gliomas, determining how co-occurring mutations influence resistance, and evaluating novel combination therapies both in vitro and in vivo.

      Conclusions

      With the genomics revolution, development of models that faithfully recapitulate specific tumor subtypes have become increasingly important. The work of Wang et al
      • Wang Q.
      • Satomi K.
      • Oh J.E.
      • Hutter B.
      • Brors B.
      • Nicolle D.
      • Liu H.
      • Wolf S.
      • Wiestler O.
      • Kleihues P.
      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      thus opens the door to ENU-induced rat gliomas, firmly placing them as a viable model for BRAF-mutant human gliomas.
      • Wang Q.
      • Satomi K.
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      • Hutter B.
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      • Liu H.
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      • Koelsch B.
      • Kindler-Röhrborn A.
      • Ohgaki H.
      Braf mutations initiate the development of rat gliomas induced by postnatal exposure to n-ethyl-n-nitrosourea.
      These ENU-induced gliomas will provide a platform for improved preclinical modeling and drug development.

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      Linked Article

      • Braf Mutations Initiate the Development of Rat Gliomas Induced by Postnatal Exposure to N-Ethyl-N-Nitrosourea
        The American Journal of PathologyVol. 186Issue 10
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          A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T->A mutations in the transmembrane domain of the Neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas remains enigmatic. We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one.
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