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BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas.

ABSTRACT: The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

SUBMITTER: Pfister S

PROVIDER: S-EPMC2289793 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Duplication of 7q34 in pediatric low-grade astrocytomas detected by high-density single-nucleotide polymorphism-based genotype arrays results in a novel BRAF fusion gene.

Project description:In the present study, DNA from 28 pediatric low-grade astrocytomas was analyzed using Illumina HumanHap550K single-nucleotide polymorphism oligonucleotide arrays. A novel duplication in chromosome band 7q34 was identified in 17 of 22 juvenile pilocytic astrocytomas and three of six fibrillary astrocytomas. The 7q34 duplication spans 2.6 Mb of genomic sequence and contains approximately 20 genes, including two candidate tumor genes, HIPK2 and BRAF. There were no abnormalities in HIPK2, and analysis of two mutation hot-spots in BRAF revealed a V600E mutation in only one tumor without the duplication. Fluorescence in situ hybridization confirmed the 7q34 copy number change and was suggestive of a tandem duplication. Reverse transcription polymerase chain reaction-based sequencing revealed a fusion product between KIAA1549 and BRAF. The predicted fusion product includes the BRAF kinase domain and lacks the auto-inhibitory N-terminus. Western blot analysis revealed phosphorylated mitogen-activated protein kinase (MAPK) protein in tumors with the duplication, consistent with BRAF-induced activation of the pathway. Further studies are required to determine the role of this fusion gene in downstream MAPK signaling and its role in development of pediatric low-grade astrocytomas.

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Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer.

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| S-EPMC539203 | biostudies-literature

A brain-penetrant RAF dimer antagonist for the noncanonical BRAF oncoprotein of pediatric low-grade astrocytomas.

Project description:BackgroundActivating 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.MethodsA 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.ResultsWe 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.ConclusionMLN2480 may be an effective therapeutic for BRAF mutant pediatric astrocytomas.

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Advances in the treatment of BRAF-mutant low-grade glioma with MAPK inhibitors.

Project description: Not available

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Polymorphous Low-Grade Neuroepithelial Tumor of the Young (PLNTY): Molecular Profiling Confirms Frequent MAPK Pathway Activation.

Project description:Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5-52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

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Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas.

Project description:Brain tumors are the most common solid tumors of childhood, and pilocytic astrocytomas (PA) are the most common central nervous system tumor in 5 to 19 year olds. Little is known about the genetic alterations underlying their development. Here, we describe a tandem duplication of approximately 2 Mb at 7q34 occurring in 66% of PAs. This rearrangement, which was not observed in a series of 244 higher-grade astrocytomas, results in an in-frame fusion gene incorporating the kinase domain of the BRAF oncogene. We further show that the resulting fusion protein has constitutive BRAF kinase activity and is able to transform NIH3T3 cells. This is the first report of BRAF activation through rearrangement as a frequent feature in a sporadic tumor. The frequency and specificity of this change underline its potential both as a therapeutic target and as a diagnostic tool.

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A new GTF2I-BRAF fusion mediating MAPK pathway activation in pilocytic astrocytoma.

Project description:Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. A recurrent feature of PA is deregulation of the mitogen activated protein kinase (MAPK) pathway most often through KIAA1549-BRAF fusion, but also by other BRAF- or RAF1-gene fusions and point mutations (e.g. BRAFV600E). These features may serve as diagnostic and prognostic markers, and also facilitate development of targeted therapy. The aims of this study were to characterize the genetic alterations underlying the development of PA in six tumor cases, and evaluate methods for fusion oncogene detection. Using a combined analysis of RNA sequencing and copy number variation data we identified a new BRAF fusion involving the 5' gene fusion partner GTF2I (7q11.23), not previously described in PA. The new GTF2I-BRAF 19-10 fusion was found in one case, while the other five cases harbored the frequent KIAA1549-BRAF 16-9 fusion gene. Similar to other BRAF fusions, the GTF2I-BRAF fusion retains an intact BRAF kinase domain while the inhibitory N-terminal domain is lost. Functional studies on GTF2I-BRAF showed elevated MAPK pathway activation compared to BRAFWT. Comparing fusion detection methods, we found Fluorescence in situ hybridization with BRAF break apart probe as the most sensitive method for detection of different BRAF rearrangements (GTF2I-BRAF and KIAA1549-BRAF). Our finding of a new BRAF fusion in PA further emphasis the important role of B-Raf in tumorigenesis of these tumor types. Moreover, the consistency and growing list of BRAF/RAF gene fusions suggests these rearrangements to be informative tumor markers in molecular diagnostics, which could guide future treatment strategies.

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Correction: A new GTF2I-BRAF fusion mediating MAPK pathway activation in pilocytic astrocytoma.

Project description:[This corrects the article DOI: 10.1371/journal.pone.0175638.].

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DNA methylation analysis of paediatric low-grade astrocytomas identifies a tumour-specific hypomethylation signature in pilocytic astrocytomas.

Project description:Low-grade gliomas (LGGs) account for about a third of all brain tumours in children. We conducted a detailed study of DNA methylation and gene expression to improve our understanding of the biology of pilocytic and diffuse astrocytomas. Pilocytic astrocytomas were found to have a distinctive signature at 315 CpG sites, of which 312 were hypomethylated and 3 were hypermethylated. Genomic analysis revealed that 182 of these sites are within annotated enhancers. The signature was not present in diffuse astrocytomas, or in published profiles of other brain tumours and normal brain tissue. The AP-1 transcription factor was predicted to bind within 200 bp of a subset of the 315 differentially methylated CpG sites; the AP-1 factors, FOS and FOSL1 were found to be up-regulated in pilocytic astrocytomas. We also analysed splice variants of the AP-1 target gene, CCND1, which encodes cell cycle regulator cyclin D1. CCND1a was found to be highly expressed in both pilocytic and diffuse astrocytomas, but diffuse astrocytomas have far higher expression of the oncogenic variant, CCND1b. These findings highlight novel genetic and epigenetic differences between pilocytic and diffuse astrocytoma, in addition to well-described alterations involving BRAF, MYB and FGFR1.

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