Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma

Abstract

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gainof-function mutations. To investigate the mutation spectrum within the proto-oncogene encoding the Ser/Thr-kinase B-Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well-known BRAF V600E mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAF V600E mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras-dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B-Raf insT ) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ ERK activation potential comparable to those of B-Raf V600E . Notably, replacement of threonines by valine residues had similar effects on B-Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B-Raf insT and B-Raf V600E , but not B-Raf wt , provoke drastic morphological alterations in human astrocytes.B-Raf plays a pivotal role in the activation of the Ras/Raf/ MEK/ERK pathway, which controls cellular proliferation, survival and differentiation. 1,2 B-Raf is highly expressed in neuronal tissues and is involved in various brain functions.3 A key event in B-Raf activation is its interaction with activated Ras (Ras-GTP) and the associated phosphorylation of the LAT 599 VKS 602 -motif within the activation segment, which is encoded by exon 15. 4 Phosphorylation of T599 and S602 reorientates the activation segment and induces full kinase activity.5 Structural analyses revealed that, in inactive B-Raf, a hydrophobic interaction between F468 of the glycine-rich P-loop and V600 within the activation segment orientates the ATP-coordinating DFG motif to a position that is incompatible with catalysis.5 Upon LATVKS-motif phosphorylation, however, this hydrophobic interaction is disrupted, and the DFG motif is reorientated in such a manner that an efficient phosphotransferase reaction can occur.Dysregulated B-Raf signaling is of growing importance in oncology and is currently being targeted through the development and clinical application of B-Raf and MEK inhibitors.

Novel BRAF mutation in melanoma: A case report

“…BRAF mutational status of the primary lesion was assessed by both Sanger sequencing and pyrosequencing, using the Anti-EGFR MoAb response BRAF Status kit (Diatech S.p.A., Jesi, Italy). Genetic analysis showed a rare aminoacidic insertion in codon 599 of the BRAF gene (1797_1798insACA, T599insT: Although in this study and in cited reports (8,(10)(11)(12)(13)(14) all mutations lead to the same T599dup alteration at amino acid level, at DNA level they are described as an ACA duplication (11,12), and as an insertion of TAC between C and A in the other studies. In silico analysis, performed on BRAFV599ins and BRAFT599insT (9,12), indicates that mutations involving the P-loop make the active geometrical conformation more stable than the wild-type counterpart, causing an highly productive catalytic state.…”

“…In vitro kinase assays carried out on BRAFV599ins and BRAFV600E (9) revealed a three-to-five-fold increase in the enzymatic activity (BRAF-induced phosphorylation of MEK and MAPK) of both mutants compared to BRAFWT. Similarly, evaluation of BRAFT599insT (8,13) confirmed the tumorigenic potential of this mutation. The two cited TKIs are not currently employed in treatment of tumors other than advanced and metastatic melanoma.…”

“…Herein, we report a single case of primary cutaneous melanoma showing a mutation occurring in the P-loop activating site (c.1797_1798insACA, T599insT), which was not previously described in melanomas, but only rarely found in Pilocytic Astrocytoma (PA), Papillary Thyroid Carcinoma (PTC) and Anaplastic Thyroid Carcinoma (ATC) (8)(9)(10)(11). In silico and in vitro data indicate that rare and/or complex mutations in codons 599-601 increase kinase activity similarly to the typical V600E (8,9,12,13).…”

Epilepsy Related to Developmental Tumors and Malformations of Cortical Development

“…1d), desmoplastic infantile GGs [38], and DNTs [31,39]. The presence of the BRAF V600E mutation in DNTs strongly supports a relationship between DNTs and GGs, pointing to the pathogenic role of BRAF in different entities within the large spectrum of GNT, including other low-grade tumors arising in young age groups, such as pilocytic astrocytomas and pleomorphic xanthoastrocytoma [36,40]. Interestingly, the BRAF V600E mutation has been shown to be associated with the expression of phosphorylated ribosomal S6 protein (pS6; marker of mTOR pathway activation) in GNT [31].…”

Pilocytic astrocytomas of the optic nerve and their relation to pilocytic astrocytomas elsewhere in the central nervous system

“…[8][9][10][11][12][13] The biologic significance of BRAF duplication lies in the activation of the MAPK pathway, which can drive tumor proliferation. 9 In addition to KIAA1549:BRAF fusion product, other molecular alterations have been reported in pilocytic astrocytoma, including other BRAF fusion products, 14,15 rare BRAF V600E mutations, 16 BRAF insertions, 17 and KRAS mutations. 18 This list further highlights the critical importance of the MAPK pathway in these tumors.…”