Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma

Abstract

Pilocytic astrocytomas (PAs), WHO malignancy grade I, are the most frequently occurring central nervous system tumour in 5- to 19-year-olds. Recent reports have highlighted the importance of MAPK pathway activation in PAs, particularly through a tandem duplication leading to an oncogenic BRAF fusion gene. Here, we report two alternative mechanisms resulting in MAPK activation in PAs. Firstly, in striking similarity to the common BRAF fusion, tandem duplication at 3p25 was observed, which produces an in-frame oncogenic fusion between SRGAP3 and RAF1. This fusion includes the Raf1 kinase domain, and shows elevated kinase activity when compared with wild-type Raf1. Secondly, a novel 3 bp insertion at codon 598 in BRAF mimics the hotspot V600E mutation to produce a transforming, constitutively active BRaf kinase. Although these two alterations are not common, they bring the number of cases with an identified 'hit' on the Ras/Raf-signalling pathway to 36 from our series of 44 (82%), confirming its central importance to the development of pilocytic astrocytomas.

Figure 1. Identification and characterisation of a novel RAF1 fusion gene

(a) An array-comparative genomic hybridisation (aCGH) plot of PA20 showing gain between clones RP11-334L22 and RP11-163D23 at 3p25. The array has been described previously (Jones et al., 2006). Raw data have been deposited with the Gene Expression Omnibus (GEO), accession no. GSE11263. (b) RT-PCR with primers in SRGAP3 exon 8 (PC5536) and RAF1 exon 10 (PC5537) give a 230bp product with cDNA from PA20, but not with cDNA from PA44 (lacking 3p25 gain) or normal brain (NB. Ambion, Austin, TX). A control PCR with primers in RAF1 exons 7 and 10 (PC5630 and PC5537, respectively) gave a 330bp product from wild-type RAF1 in all three samples. M; 100bp ladder (Invitrogen, Paisley, UK), –ve; no template control. (c) A sequence trace confirming a fusion between SRGAP3 exon 12 and RAF1 exon 10. RT-PCR product was cycle-sequenced with PC5536 using BigDye v3.1 chemistry and run on a 3100-Avant Genetic Analyser (Applied Biosystems, Cheshire, UK). Primer sequences and PCR conditions are given in Supplementary Table 2. The fusion transcript sequence has been deposited in the EMBL Nucleotide Sequence Database, accession no. FM209427. (d) Schematic representation of the tandem duplication rearrangement, and the SRGAP3:RAF1 fusion protein. FCH; Fes/CIP4 homology domain, KD; kinase domain. (e) NIH3T3 cells were stably retrovirally transduced as previously described (Jones et al., 2008) with either empty pBabe-puro vector (EV), wild-type RAF1 (WT), or the SRGAP3:RAF1 fusion gene (S:R). Protein was extracted from cells lysed in RIPA buffer supplemented with Complete protease inhibitor cocktail (Roche Diagnostics, Burgess Hill, UK), run on a 4-12% NuPage gel and transferred to a 45μm PVDF membrane (Invitrogen). Membranes were blocked for 1hr with 5% milk in TBS/0.1% Tween-20 and probed with either anti-phospho-MEK1/2 (top panel, rabbit monoclonal, Cell Signalling Technology, Danvers, MA) or anti-MEK1/2 loading control (bottom panel, rabbit polyclonal, Cell Signalling Technology). Goat HRP-conjugated anti-Rabbit Ig secondary antibody was used. Blots were visualised with ECL+ (GE Healthcare, Little Chalfont, UK) using a Fujifilm LAS-4000 imager (Fujifilm UK Ltd, Bedford, UK). Expression from the transduced constructs was demonstrated by probing with anti-HA (see Supplementary Figure 1). Cells expressing the srGAP3:Raf1 fusion protein (S:R) show elevated endogenous phospho-MEK levels compared with those expressing Raf1^WT (WT) or transduced with empty vector (EV). Two independent transductions gave similar results. -ve; lysis buffer only. (f) The same stably transduced cells were assayed for growth in soft agarose as previously described (Jones et al., 2008). Three wells per construct were plated using 10,000 cells/well. Representative fields are shown following 14 days of growth. Cells expressing the srGAP3:Raf1 fusion protein demonstrated a transformed phenotype as evidenced by their anchorage-independent growth. Scale bars = 250μm. (g) Colonies greater than 0.1mm were scored for each well. The mean score for each construct is given with error bars indicating +/− 1SD. ***; p<0.0005, two-tailed Student’s t-Test.

Figure 2. Identification and functional characterisation of a novel BRAF mutation

(a) Sample PA12 shows a complex sequence trace due to a 3bp insertion in BRAF exon 15. This exon was then amplified from PA12 tumour and blood genomic DNA with primers PC4580 and PC4581, and cloned into a TOPO-TA vector (Invitrogen). Fourteen colonies for each were assessed by cycle-sequencing as described above using a T3 primer. Nine of the tumour colonies showed the trinucleotide insertion (bottom right), which was not seen in any of the blood DNA clones (top right). (b) Kinase activity of wild-type (WT), V600E and ins598T (insT) BRaf was assayed with an in vitro kinase assay kit as previously described (Jones et al., 2008). BRaf^ins598T showed constitutive kinase activity at a similar level to BRaf^V600E, both of which were significantly more active than BRaf^WT. Each protein was assayed in triplicate. Two independent transfections showed similar results. (c) NIH3T3 cells were retrovirally transduced with either pBabe-puro vector (EV), wild-type BRAF (WT), BRAF^V600E (V600E), or BRAF^ins598T (insT). Expression from the transduced constructs was demonstrated by western blotting with anti-HA (as described above, see Supplementary Figure 2). Stably transduced cells were then assayed for growth in soft agarose as previously described (Jones et al., 2008). Three wells per construct were plated using 10,000 cells/well. Representative fields are shown following 11 days of growth. Cells expressing mutant BRaf demonstrated a transformed phenotype as evidenced by their anchorage-independent growth. Scale bars = 250μm. (d) Colonies greater than 0.1mm were scored for each well. The mean score for each construct is given with error bars indicating +/− 1SD. *; p<0.05, **; p<0.005, ***; p<0.0005, two-tailed Student’s t-Test.

Figure 3. An overview of MAPK pathway alterations in PAs

The frequency of each of the activating alterations in the Ras/Raf pathway observed in our tumour series is indicated (n=44). RTK; receptor tyrosine kinase, TF; transcription factor.