<i>NTRK1</i> Fusion in Glioblastoma Multiforme

<div><p>Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3′ exons of neurotrophic tyrosine kinase receptor type 1 (<i>NTRK1</i>, encoding TrkA) are fused to 5′ exons of the genes that are highly expressed in neuronal tissues, neurofascin (<i>NFASC</i>) and brevican (<i>BCAN</i>). The fusions preserved both the transmembrane and kinase domains of <i>NTRK1</i> in frame. <i>NTRK1</i> is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked <i>NTRK1</i> expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated <i>NTRK1</i>-pathway activity. Lentiviral transduction of the <i>NFASC-NTRK1</i> fusion gene in NIH 3T3 cells increased proliferation <i>in vitro</i>, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.</p></div

<i>NFASC-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage (expression) of genomic regions along <i>NFASC</i> and <i>NTRK1</i>. The dotted line marks the DNA-level break-points in the two genes, as instructed by the fusion-point mapping result in panel B. (<b>B</b>) A schematic of pre-mRNAs of the <i>NFASC-NTRK1</i> fusion gene. Top and bottom sequences in black are the reads that map onto the DNA-level fusion-point. The fusion-point is mapped with slight ambiguity due to 2-nt-long micro-homology between the two break-points in the involved genes. (<b>C</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p

Molecular consequences of <i>NTRK1</i>-fusion.

<p>(<b>A</b>) <i>NTRK1</i> expression in 170 TCGA GBM samples (from 162 patients) with RNA-Seq data. Samples bearing <i>NTRK1</i>-fusion genes are marked and labeled. (<b>B</b>) Relationship between <i>NTRK1</i> expression and NGF/TrkA-downstream pathway activity in 526 TCGA GBM samples (from 526 patients) with microarray gene expression data. Samples with <i>NTRK1</i>-fusion are marked with red circles. Two other samples with outlier <i>NTRK1</i> expression are marked with blue circles (TCGA-32-4209, TCGA-19-5947).</p

<i>BCAN-NTRK1</i> fusion.

<p>(<b>A</b>) Per-nucleotide read coverage of genomic regions along <i>BCAN</i> and <i>NTRK1</i>. The dotted line marks approximate positions where the fusion has occurred. (<b>B</b>) A schematic of spliced transcripts of the fusion gene. Bottom sequences in black are the reads that map onto the chimeric exon-exon splicing junction.</p

Tumorigenic activities of <i>NFASC-NTRK1</i> fusion gene.

<p>(<b>A</b>) <i>NFASC-NTRK1</i> and <i>EGFR</i> vIII mRNA expression in NIH 3T3 cells, determined by RT-PCR. (<b>B</b>) Proliferation of NIH 3T3 cells lentivirally infected with the indicated viruses. Error bars are 95% confidence intervals. (<b>C</b>) Number of colonies in a unit microscopic field, formed by NIH 3T3 cells infected with the indicated viruses. Red lines are the average within each group. (<b>D</b>) Morphology of individual colonies in soft agar, formed by NIH 3T3 cells infected with the indicated viruses. (<b>E</b>) Incidences of subcutaneous tumor formation in the mice injected with NIH 3T3 cells infected with the indicated viruses. (<b>F</b>) Inhibition of proliferation by three independent shRNAs targeting the <i>NTRK1</i> fusion transcripts. Error bars are standard deviations of five-replicate experiments.</p

Top-20 potential gene fusions predicted by discordant read pair analysis.

a<p>Fusion type: intra, intra-chromosomal; inter, inter-chromosomal; read-through, the involved genes are adjacent and on the same strand; cis, the involved genes are adjacent and on the opposite strands.</p>b<p>For the fusions that were not excluded by indicated reasons, gene 1 and gene 2 correspond to the 5′- or 3′-partner of each fusion.</p>c<p>Sample IDs are abbreviated.</p>d<p>RESPER is FusionSeq-reported scores for prioritization. The fusions that were not excluded are indicated in bold font.</p

Effect of resveratrol on islet fibrosis.

<p>(A) Representative Masson’s trichrome staining from pancreatic tissue (x 200). (B) Quantification of islet fibrosis. Data are the mean ± SE. *<i>P</i><0.05 vs. vehicle-treated db-control group. n = 8 in each group.</p

Effect of oral administration of resveratrol on the area under the curve of glucose (AUCg) during a glucose tolerance test and insulin tolerance test.

<p>(A) AUCg during 2h glucose tolerance test. (B) K<i>itt</i> values (%/min) from the insulin tolerance test at 12 weeks as a surrogate insulin sensitivity index. Data are the mean ± SE. n = 8 in each group. *<i>P</i><0.05 vs. vehicle-treated control group.</p

Effects of long-term resveratrol treatment on pancreas weight.

<p>Data are the mean ± SE. n = 8 in each group. *<i>P</i><0.05 vs. vehicle-treated control group.</p

Measurement of urinary ROS marker generation.

<p>Values are given as the mean ± SE for groups (n = 8 for each). *<i>P</i><0.005 vs. dm-control; **<i>P</i><0.05 vs. db-control group.</p