Identification of recurrent NAB2-STAT6 gene fusions in solitary fibrous tumor by integrative sequencing

Abstract

A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole-exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs identified the presence of a NAB2-STAT6 gene fusion in all tumors. Using RT-PCR and sequencing, we detected this fusion in all 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated the expression of EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.

Figure 1

Integrative sequencing and mutational analysis of patient MO_1005 (SFT index case). (a) CT image of the biopsied liver metastasis (arrow). Arrow indicates metastasis that was biopsied. Scale bar equals 10 cm. (b) Hematoxylin and eosin staining of index case. Scale bar, 200 μm. (c) Hematoxylin and eosin staining of index case. Scale bar, 100 μm. Please note, b and c differ only in magnification. (d) Immunoreactivity for CD34. Scale bar, 100 μm. Images in a–d were all produced from a liver biopsy sample from the index case. (e) Gene copy number landscape of the index case as assessed by whole-exome sequencing of the tumor to germline sequence. The arrow indicates copy loss at the STAT6 locus. (f) Schematic of the NAB2-STAT6 gene fusion detected in the index case by paired-end transcriptome sequencing. The hashed region indicates exons not shown.

Figure 2

Validation and recurrence of NAB2-STAT6 gene fusions in SFT. (a) RT-PCR and capillary sequencing of the index case and additional SFT cases using primers for NAB2 exon 6 and STAT6 exon 19. The sequencing trace of the index case (right) shows the chimeric junction between NAB2 and STAT6. (b) Genomic long-range PCR of the index case confirming the existence of the NAB2-STAT6 gene fusion at the DNA level. Gel electrophoresis of the amplified product (left, arrow) and schematic of exon-intron structure of the index NAB2-STAT6 gene fusion (right) are shown. (c) Schematics of additional NAB2-STAT6 gene fusions identified by transcriptome sequencing of 6 SFT samples.

Figure 3

Characterization and functional analysis of the NAB2-STAT6 fusion protein. (a) Schematics of the predicted NAB2-STAT6 fusion protein products identified in this study. EBD, EGR1-binding domain; NCD2, NAB2 conserved domain; RD, transcriptional repressor domain; SH2, Src homology 2; CCD1, coiled-coil domain 1; DBD, DNA-binding domain; TAD, transcriptional activator domain. (b) Immunoblot analysis of three SFT tumors (T) and matched normal tissue (N) employing an antibody against a C-terminal epitope of STAT6, which is found in all the NAB2-STAT6 fusions thus far identified. SFT-14 tumor expresses the larger 150-kDa NAB2-STAT6 fusion protein. SFT-10 and SFT-44 tumors both express the smaller, more common 90-kDa NAB2-STAT6 fusion. MW, molecular weight; WT, wild type. Arrowheads indicate NAB2-STAT6 fusion proteins. (c) Immunofluorescence using the antibody in b, suggesting nuclear localization (red) of the NAB2-STAT6 protein in a representative SFT case. Scale bar, 50 μm. (d–f) Stable RWPE-1 cell line pools expressing low and high levels of Flag-tagged NAB2-STAT6 (MO_1005 fusion structure) were generated. (d) Immunoblot analysis with an antibody to Flag. (e) Cell proliferation assays carried out with live-cell imaging. Data are shown as cell confluence versus time at 3-h intervals. Each data point is the mean of quadruplicate measurements. (f) qRT-PCR for EGR1 target genes IGF2, H19 and RRAD. Results were normalized to GAPDH levels. Means of triplicates with the s.d. are shown.

Figure 4

A proposed model for the function of the NAB2-STAT6 gene fusion in SFT. (a) NAB2-STAT6 fusion protein enhances EGR1-induced promoter activity. We transfected 293T cells with vectors expressing the indicated proteins along with a constitutively expressed Renilla luciferase construct and one of the two reporter constructs EGR1-responsive firefly luciferase reporter or STAT6-responsive firefly luciferase reporter. Cell lysates were prepared 24 h after transfection and measured for EGR1 and STAT6 activity. Means of quadruplicates with the s.d. are shown. (b) ChIP identified the recruitment of NAB2-STAT6 fusion protein to the promoters of EGR1-responsive genes. We transfected 293T cells with only vector expressing EGR1 (C) or cotransfected with vectors expressing EGR1 and Flag-NAB2-STAT6 (F). Crosslinked DNA-protein lysates were immunoprecipitated with antibody to Flag. The promoter fragments containing consensus EGR1-binding sites were PCR amplified from both samples. Input samples were loaded as a control. IGX1A primers that detect a specific genomic DNA sequence within an ORF-free intergenic region were used as a negative control. Two distinct EGR1-binding sites in NAB2 and CASP9 are shown. (c) Outlier gene expression in SFT predicted to be the result of NAB2-STAT6–driven constitutive activation of EGR1-mediated pathways. Data from RNA-seq analysis of 7 SFTs relative to a compendium of 282 previously sequenced tumor tissues are shown (Supplementary Table 6). RPKPM (reads per kilobase per million reads) values for the compendium are summarized by box-and-whisker plots showing the minimum, quartile 1, median, quartile 3 and maximum observed expression levels across the compendium. RPKPM values for the seven SFT samples are shown in red. (d) Model of the NAB2 and EGR1 signaling loops. RD, repressor domain; AD, activation domain.