RESUMO
The basic helix-loop-helix transcription factors oligodendrocyte transcription factor 1 (OLIG1) and OLIG2 are structurally similar and, to a first approximation, coordinately expressed in the developing CNS and postnatal brain. Despite these similarities, it was apparent from early on after their discovery that OLIG1 and OLIG2 have non-overlapping developmental functions in patterning, neuron subtype specification and the formation of oligodendrocytes. Here, we summarize more recent insights into the separate roles of these transcription factors in the postnatal brain during repair processes and in neurological disease states, including multiple sclerosis and malignant glioma. We discuss how the unique functions of OLIG1 and OLIG2 may reflect their distinct genetic targets, co-regulator proteins and/or post-translational modifications.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Animais , Encéfalo/citologia , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/terapia , Neurônios/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Células 3T3 , Alelos , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Estudos de Coortes , Hibridização Genômica Comparativa , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Família Multigênica , Mutação , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
The bHLH transcription factor Olig2 is expressed in cycling neural progenitor cells but also in terminally differentiated, myelinating oligodendrocytes. Sustained expression of Olig2 is counterintuitive because all known functions of the protein in expansion of neural progenitors and specification of oligodendrocyte progenitors are completed with the formation of mature white matter. How are the biological functions of Olig2 suppressed in terminally differentiated oligodendrocytes? In previous studies, we have shown that a triple serine motif in the amino terminus of Olig2 is phosphorylated in cycling neural progenitors but not in their differentiated progeny. We now show that phosphorylation of the triple serine motif regulates intranuclear compartmentalization of murine Olig2. Phosphorylated Olig2 is preferentially localized to a transcriptionally active "open" chromatin compartment together with coregulator proteins essential for regulation of gene expression. Unphosphorylated Olig2, as seen in mature white matter, is localized mainly within a transcriptionally inactive, chromatin fraction characterized by condensed and inaccessible DNA. Of special note is the observation that the p53 tumor suppressor protein is confined to the open chromatin fraction. Proximity ligation assays show that phosphorylation brings Olig2 within 30 nm of p53 within the open chromatin compartment. The data thus shed light on previously noted promitogenic functions of phosphorylated Olig2, which reflect, at least in part, an oppositional relationship with p53 functions.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Núcleo Celular/química , Núcleo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/química , Células-Tronco Neurais/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Núcleo Celular/genética , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fator de Transcrição 2 de Oligodendrócitos , Fosforilação/genética , GravidezRESUMO
Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.
Assuntos
Sistema Urogenital/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Rim/metabolismo , CamundongosRESUMO
TGFbeta functions as a tumor suppressor in some contexts and a tumor promoter in others. In a recent issue of Cancer Cell, Bruna et al. (2007) shed light on an epigenetic mechanism that underlies this schizophrenic behavior in malignant glioma. Their findings highlight a stem cell/cancer link...and a potential blind spot in large-scale cancer genome sequencing projects.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Proteínas Smad/genética , Fator de Crescimento Transformador beta/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Epigênese Genética/genética , Glioma/metabolismo , Glioma/fisiopatologia , Humanos , Proteínas Smad/metabolismo , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Gliomas consist of multiple histologic and molecular subtypes with different clinical phenotypes and responsiveness to treatment. However, enrollment criteria for clinical trials still largely do not take into account these underlying molecular differences. We have incorporated a high-throughput tumor genotyping program based on the ABI SNaPshot platform as well as other molecular diagnostic tests into the standard evaluation of glioma patients in order to assess whether prospective molecular profiling would allow rational patient selection onto clinical trials. From 218 gliomas we prospectively collected SNaPshot genotyping data on 68 mutated loci from 15 key cancer genes along with data from clinical assays for gene amplification (EGFR, PDGFRA, MET), 1p/19q co-deletion and MGMT promoter methylation. SNaPshot mutations and focal gene amplifications were detected in 38.5 and 47.1 % of glioblastomas, respectively. Genetic alterations in EGFR, IDH1 and PIK3CA closely matched frequencies reported in recent studies. In addition, we identified events that are rare in gliomas although are known driver mutations in other cancer types, such as mutations of AKT1, BRAF and KRAS. Patients with genetic alterations that activate signaling pathways were enrolled onto genetically selective clinical trials for malignant glioma as well as for other solid cancers. High-throughput molecular profiling incorporated into the routine clinical evaluation of glioma patients may enable the rational selection of patients for targeted therapy clinical trials and thereby improve the likelihood that such trials succeed.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Perfilação da Expressão Gênica , Genótipo , Humanos , Hibridização in Situ Fluorescente , Biologia Molecular/métodos , Mutação , Reação em Cadeia da PolimeraseRESUMO
In this issue of Neuron, Jackson et al. show that adult neural stem cells (B cells) express PDGF receptors. Functional analysis of PDGF signaling in these neural progenitors resonates in provocative ways with an older body of literature on PDGF autocrine loops in malignant glioma and with more recent observations on the bHLH transcription factor Olig2.
Assuntos
Glioma/metabolismo , Glioma/patologia , Neurônios/metabolismo , Neurônios/patologia , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Adulto , HumanosRESUMO
Forward genetic screens in genetically accessible invertebrate organisms such as Drosophila melanogaster have shed light on transcription factors that specify formation of neurons in the vertebrate CNS. However, invertebrate models have, to date, been uninformative with respect to genes that specify formation of the vertebrate glial lineages. All recent insights into specification of vertebrate glia have come via monitoring the spatial and temporal expression patterns of individual transcription factors during development. In studies described here, we have taken this approach to the genome scale with an in silico screen of the Mahoney pictorial atlas of transcription factor expression in the developing CNS. From the population of 1445 known or probable transcription factors encoded in the mouse genome, we identify 12 novel transcription factors that are expressed in glial lineage progenitor cells. Entry-level screens for biological function establish one of these transcription factors, Klf15, as sufficient for genesis of precocious GFAP-positive astrocytes in spinal cord explants. Another transcription factor, Tcf4, plays an important role in maturation of oligodendrocyte progenitors.
Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica , Testes Genéticos , Genoma/fisiologia , Neuroglia/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Testes Genéticos/métodos , Camundongos , Camundongos Knockout , Neuroglia/citologia , Gravidez , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.
Assuntos
Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Células-Tronco Neurais/citologia , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Neoplasias Encefálicas/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Microglia/patologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligodendroglia/citologia , Proteínas de Fusão Oncogênica/metabolismo , Células Tumorais CultivadasRESUMO
In a widely held view of vertebrate CNS development, neurons, astrocytes, and oligodendrocytes arise from a common tripotent progenitor cell. However, tripotent progenitors have never been detected in developing embryos. In this issue of Neuron, Gabay et al. show that tripotent progenitors can be created in vitro by deregulation of normal dorsoventral positional cues.
Assuntos
Diferenciação Celular/fisiologia , Sistema Nervoso Central/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Astrócitos/metabolismo , Padronização Corporal/fisiologia , Agregação Celular , Técnicas de Cultura de Células , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Proteínas Hedgehog , Camundongos , Modelos Neurológicos , Oligodendroglia/metabolismo , Ratos , Transativadores/metabolismoRESUMO
Transcription factors with bHLH motifs modulate critical events in the development of the mammalian neocortex. Multipotent cortical progenitors are maintained in a proliferative state by bHLH factors from the Id and Hes families. The transition from proliferation to neurogenesis involves a coordinate increase in the activity of proneural bHLH factors (Mash1, Neurogenin1, and Neurogenin2) and a decrease in the activity of Hes and Id factors. As development proceeds, inhibition of proneural bHLH factors in cortical progenitors promotes the formation of astrocytes. Finally, the formation of oligodendrocytes is triggered by an increase in the activity of bHLH factors Olig1 and Olig2 that may be coupled with a decrease in Id activity. Thus, bHLH factors have key roles in corticogenesis, affecting the timing of differentiation and the specification of cell fate.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Sequências Hélice-Alça-Hélice/fisiologia , Neocórtex/citologia , Neocórtex/embriologia , Animais , Astrócitos/citologia , Humanos , Células-Tronco Multipotentes/fisiologia , Neurônios/citologia , Oligodendroglia/citologia , Fatores de Transcrição/química , Fatores de Transcrição/fisiologiaRESUMO
Primary brain tumors consist of neoplasms with varied molecular defects, morphologic phenotypes, and clinical outcomes. The genetic and signaling abnormalities involved in tumor initiation and progression of the most prevalent adult primary brain tumors, including gliomas, meningiomas, and medulloblastomas, are described in this article. The current understanding of the cell-of-origin of these neoplasms is reviewed, which suggests that the malignant phenotype is propelled by cells with stem-like qualities. A comprehensive understanding of the molecular basis of transformation and the cell-of-origin of these neoplasms will enable the formulation of more targeted treatment alternatives that could improve survival and quality of life.
Assuntos
Neoplasias Encefálicas/patologia , Células-Tronco/patologia , Células-Tronco/fisiologia , Adulto , Neoplasias Encefálicas/genética , Genes p53/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fenótipo , RNA Mensageiro/genética , Transdução de SinaisRESUMO
Pheochromocytomas are catecholamine-secreting tumors that result from mutations of at least six different genes as components of distinct autosomal dominant disorders. However, there remain familial occurrences of pheochromocytoma without a known genetic defect. We describe here a familial pheochromocytoma syndrome consistent with digenic inheritance identified through a combination of global genomics strategies. Multipoint parametric linkage analysis revealed identical LOD scores of 2.97 for chromosome 2cen and 16p13 loci. A two-locus parametric linkage analysis produced maximum LOD score of 5.16 under a double recessive multiplicative model, suggesting that both loci are required to develop the disease. Allele-specific loss of heterozygosity (LOH) was detected only at the chromosome 2 locus in all tumors from this family, consistent with a tumor suppressor gene. Four additional pheochromocytomas with a similar genetic pattern were identified through transcription profiling and helped refine the chromosome 2 locus. High-density LOH mapping with single nucleotide polymorphism-based array identified a total of 18 of 62 pheochromocytomas with LOH within the chromosome 2 region, which further narrowed down the locus to <2 cM. This finding provides evidence for two novel susceptibility loci for pheochromocytoma and adds a recessive digenic trait to the increasingly broad genetic heterogeneity of these tumors. Similarly, complex traits may also be involved in other familial cancer syndromes.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Feocromocitoma/genética , Adulto , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 2 , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Genômica/métodos , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
During central nervous system development, oligodendrocytes must be formed in proportion to the number of neurons requiring their services. In this issue of Neuron, Voronova et al. (2017) show how cortical interneurons modulate oligodendrogenesis through a cytokine-mediated paracrine interaction.
Assuntos
Interneurônios , Controle da População , Neurônios , OligodendrogliaRESUMO
BACKGROUND: Astrocytomas are the most common primary human brain tumors. Receptor tyrosine kinases (RTKs), including tyrosine receptor kinase B (TrkB, also known as tropomyosin-related kinase B; encoded by neurotrophic tyrosine kinase receptor type 2 [NTRK2]), are frequently mutated by rearrangement/fusion in high-grade and low-grade astrocytomas. We found that activated TrkB can contribute to the development of astrocytoma and might serve as a therapeutic target in this tumor type. METHODS: To identify RTKs capable of inducing astrocytoma formation, a library of human tyrosine kinases was screened for the ability to transform murine Ink4a-/-/Arf-/- astrocytes. Orthotopic allograft studies were conducted to evaluate the effects of RTKs on the development of astrocytoma. Since TrkB was identified as a driver of astrocytoma formation, the effect of the Trk inhibitors AZD1480 and RXDX-101 was assessed in astrocytoma cells expressing activated TrkB. RNA sequencing, real-time PCR, western blotting, and enzyme-linked immunosorbent assays were conducted to characterize NTRK2 in astrocytomas. RESULTS: Activated TrkB cooperated with Ink4a/Arf loss to induce the formation of astrocytomas through a mechanism mediated by activation of signal transducer and activator of transcription 3 (STAT3). TrkB activation positively correlated with Ccl2 expression. TrkB-induced astrocytomas remained dependent on TrkB signaling for survival, highlighting a role of NTRK2 as an addictive oncogene. Furthermore, the QKI-NTRK2 fusion associated with human astrocytoma transformed Ink4a-/-/Arf-/- astrocytes, and this process was also mediated via STAT3 signaling. CONCLUSIONS: Our findings provide evidence that constitutively activated NTRK2 alleles, notably the human tumor-associated QKI-NTRK2 fusion, can cooperate with Ink4a/Arf loss to drive astrocytoma formation. Therefore, we propose NTRK2 as a potential therapeutic target in the subset of astrocytoma patients defined by QKI-NTRK2 fusion.
Assuntos
Fator 1 de Ribosilação do ADP/fisiologia , Astrócitos/patologia , Astrocitoma/patologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptor trkB/metabolismo , Animais , Astrócitos/enzimologia , Astrocitoma/enzimologia , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
During development of the vertebrate CNS, the basic helix-loop-helix (bHLH) transcription factor Olig2 sustains replication competence of progenitor cells that give rise to neurons and oligodendrocytes. A pathological counterpart of this developmental function is seen in human glioma, wherein Olig2 is required for maintenance of stem-like cells that drive tumor growth. The mitogenic/gliomagenic functions of Olig2 are regulated by phosphorylation of a triple serine motif (S10, S13, and S14) in the amino terminus. Here, we identify a set of three serine/threonine protein kinases (glycogen synthase kinase 3α/ß [GSK3α/ß], casein kinase 2 [CK2], and cyclin-dependent kinases 1/2 [CDK1/2]) that are, collectively, both necessary and sufficient to phosphorylate the triple serine motif. We show that phosphorylation of the motif itself serves as a template to prime phosphorylation of additional serines and creates a highly charged "acid blob" in the amino terminus of Olig2. Finally, we show that small molecule inhibitors of this forward-feeding phosphorylation cascade have potential as glioma therapeutics.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Glioma/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Animais , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Glioma/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency.
Assuntos
Dosagem de Genes , Neoplasias/genética , Neoplasias/patologia , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Linhagem Celular Tumoral , HumanosRESUMO
Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.
Assuntos
Astrocitoma , Neoplasias Encefálicas , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Senescência Celular/fisiologia , Antígenos Transformantes de Poliomavirus/genética , Western Blotting , Proliferação de Células/fisiologia , Pré-Escolar , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Transcriptoma , Transdução GenéticaRESUMO
Background: Activating mutations or structural rearrangements in BRAF are identified in roughly 75% of all pediatric low-grade astrocytomas (PLGAs). However, first-generation RAF inhibitors approved for adult melanoma have poor blood-brain penetrance and are only effective on tumors that express the canonical BRAFV600E oncoprotein, which functions as a monomer. These drugs (type I antagonists that target the "DFG-in" conformation of the kinase) fail to block signaling via KIAA1549:BRAF, a truncation/fusion BRAF oncoprotein which functions as a dimer and is found in the most common form of PLGA. Methods: A panel of small molecule RAF inhibitors (including type II inhibitors, targeting the "DFG-out" conformation of the kinase) was screened for drugs showing efficacy on murine models of PLGA and on authentic human PLGA cells expressing KIAA1549:BRAF. Results: We identify a type II RAF inhibitor that serves as an equipotent antagonist of BRAFV600E, KIAA1549:BRAF, and other noncanonical BRAF oncoproteins that function as dimers. This drug (MLN2480, also known as TAK-580) has good brain penetrance and is active on authentic human PLGA cells in brain organotypic cultures. Conclusion: MLN2480 may be an effective therapeutic for BRAF mutant pediatric astrocytomas.
Assuntos
Astrocitoma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Quinases raf/antagonistas & inibidores , Animais , Astrocitoma/metabolismo , Astrocitoma/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Criança , Compostos Heterocíclicos com 3 Anéis/química , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/genética , Quinases raf/metabolismoRESUMO
BACKGROUND: Clinical genomics platforms are needed to identify targetable alterations, but implementation of these technologies and best practices in routine clinical pediatric oncology practice are not yet well established. METHODS: Profile is an institution-wide prospective clinical research initiative that uses targeted sequencing to identify targetable alterations in tumors. OncoPanel, a multiplexed targeted exome-sequencing platform that includes 300 cancer-causing genes, was used to assess single nucleotide variants and rearrangements/indels. Alterations were annotated (Tiers 1-4) based on clinical significance, with Tier 1 alterations having well-established clinical utility. OncoCopy, a clinical genome-wide array comparative genomic hybridization (aCGH) assay, was also performed to evaluate copy number alterations and better define rearrangement breakpoints. RESULTS: Cancer genomes of 203 pediatric brain tumors were profiled across histological subtypes, including 117 samples analyzed by OncoPanel, 146 by OncoCopy, and 60 tumors subjected to both methodologies. OncoPanel revealed clinically relevant alterations in 56% of patients (44 cancer mutations and 20 rearrangements), including BRAF alterations that directed the use of targeted inhibitors. Rearrangements in MYB-QKI, MYBL1, BRAF, and FGFR1 were also detected. Furthermore, while copy number profiles differed across histologies, the combined use of OncoPanel and OncoCopy identified subgroup-specific alterations in 89% (17/19) of medulloblastomas. CONCLUSION: The combination of OncoPanel and OncoCopy multiplex genomic assays can identify critical diagnostic, prognostic, and treatment-relevant alterations and represents an effective precision medicine approach for clinical evaluation of pediatric brain tumors.