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An Activated Mutant BRAF Kinase Domain Is Sufficient to Induce Pilocytic Astrocytoma in Mice


An Activated Mutant BRAF Kinase Domain Is Sufficient to Induce Pilocytic Astrocytoma in Mice

Jan Gronych et al. J Clin Invest.


Pilocytic astrocytoma (PA) is the most common type of primary brain tumor in children and the second most frequent cancer in childhood. Children with incompletely resected PA represent a clinically challenging patient cohort for whom conventional adjuvant therapies are only moderately effective. This has produced high clinical demand for testing of new molecularly targeted treatments. However, the development of new therapeutics for PA has been hampered by the lack of an adequate in vivo tumor model. Recent studies have identified activation of MAPK signaling, mainly by oncogenic BRAF activation, as a hallmark genetic event in the pathogenesis of human PA. Using in vivo retroviral somatic gene transfer into mouse neural progenitor cells, we have shown here that ectopic expression of the activated BRAF kinase domain is sufficient to induce PA in mice. Further in vitro analyses demonstrated that overexpression of activated BRAF led to increased proliferation of primary mouse astrocytes that could be inhibited by treatment with the kinase inhibitor sorafenib. Our in vivo model for PA shows that the activated BRAF kinase domain is sufficient to induce PA and highlights its role as a potential therapeutic target.


Figure 1
Figure 1. BRAF variants induce MAPK activation.
(A) Different BRAF constructs used for RCAS-mediated gene delivery. Truncated variants containing the kinase domain corresponded to exons 9–18 of the human BRAF gene. The V600E mutation is indicated. (B) Phase-contrast microscopy demonstrated altered cell morphology and focal growth of cells transduced with BRAF VE kin compared with control and other BRAF constructs. Scale bars: 100 μm.
Figure 2
Figure 2. MRI and tumor histology.
(A) Hemispheric contrast-enhancing tumor induced with BRAF VE kin, as observed in T1-weighted MRI and coronal brain sections, showed overlapping expression of GFAP, the BRAF VE kin transgene (FLAG-tag), and Erk phosphorylation after immunohistochemical staining. (B) Tumors were also induced in the brainstem and cerebellum after expression of BRAF VE kin. (C) Higher-magnification views show the presence of transgene (FLAG), tumor delineation from normal tissue, and stronger GFAP staining of tumor cells compared with adjacent normal reactive astrocytes. Nestin staining of tumor cells was low compared to endothelial cells in neoplastic and normal tissue that exhibited a strong immunoreactivity. (D) Histological comparison between human PA and murine BRAF VE kin–induced PA. H&E-stained sections showed histological features of PA, such as piloid tumor cells, eosinophilic Rosenthal fibers, and moderate cellularity. GFAP staining revealed strong immunoreactivity with a tight network of elongated processes and protein droplets characteristic for PA. The low proliferation index of both tumors was represented by Ki67 staining. Tumors displayed Erk phosphorylation in both cases, indicating constitutive MAPK activation. Scale bars: 100 μm.
Figure 3
Figure 3. Transformation and sorafenib treatment of primary astrocytes in vitro.
(A) Primary Ntv-a astrocytes were transduced in vitro with BRAF variants and GFP as a control. BRAF VE FL protein was considerably reduced, whereas all other variants showed high expression after transduction. Stronger activation of Erk phosphorylation in BRAF VE kin than in BRAF VE FL was decreased to control levels upon treatment with 5 μM sorafenib for 6 hours. DMSO was used as vehicle control. All other variants did not show increased Erk phosphorylation. BRAF transcript abundance showed high variation among constructs depending on transduction efficiency (normalized to 3 housekeeping genes [HKGs]). (B) Representative FACS plots from EdU incorporation assays of astrocytes transduced and treated as indicated, and quantification of the proliferating fraction for all BRAF variants and GFP treated with 5 μM sorafenib or DMSO control. Proliferation and RNA and protein expression were assayed from the same experiment. FSC, forward scatter. Data are mean ± SD of 3 technical replicates.

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