<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

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>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

<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

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

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

Translational Validation of Personalized Treatment Strategy Based on Genetic Characteristics of Glioblastoma

<div><p>Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by <i>in vitro</i> limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the <i>in vitro</i> clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.</p></div

Prognostic outcomes of 4 molecular subtypes of 105 SMC GBM patients.

<p>(<b>A</b>) Kaplan Meier plot shows survivals for 4 molecular subtypes of 105 SMC GBM patients, which was predicted by the NTP method. (<b>B</b>) Median overall survival lengths (Median) and 95% confidence limits (CL) of the subtypes were summarized. Log rank test was used for statistical analyses.</p

Network for GBM subtype-specific drug candidates.

<p>Eight drug candidates were directly/indirectly liked to a number of subtype-specific genes; Clomipramine: 57 proneural-specific genes; Gefitinib: 64 proneural-specific genes; Beta-Nicotinamide Adenine Dinucleotide Hydrate: 35 neural-specific genes; Bicuculline: 5 neural-specific genes; Pravastatin: 100 mesenchymal-specific genes; Resveratrol: 86 mesenchymal-specific genes; Irinotecan: 20 classical-specific genes; Paclitaxel: 79 classical-specific genes.</p

Orthotopic xenograft “AVATAR” models recapitulate the subtypes of their parental GBMs.

<p>(<b>A</b>) Predicted molecular subtypes of 25 GBM patients from the SMC GBM dataset and corresponding orthotopic xenograft “AVATAR” models were summarized. *, matched case. PN = Proneural, NL = Neural, CL = Classical, MES = Mesenchymal, ND = not-determined. (<b>B</b>) Subtype classification of 25 GBM patients by the NTP methods was compared with subtypes of corresponding orthotopic xenograft “AVATAR” models.</p