<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>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

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

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

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2

<div><p>Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both <i>in vitro</i> and <i>in vivo</i> orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM.</p></div

FoxM1 and Sox2 are upregulated in GBM cells after irradiation.

<p>(A) Western blot analysis of FoxM1 and Sox2 using the irradiated NS07-448 (left) and 387 (right) GBM cells. Cells were harvested with course of time after irradiation. β-actin was used as a loading control. (B) Representative IF images of NS07-448 GBM cells with or without irradiation using anti-FoxM1 and anti-Sox2 antibodies. Scale bar, 20 μm. (C and D) Western blot analysis of FoxM1 and Sox2 in GBM cells after irradiation. Cytoplasmic (C) and nuclear (N) fractions of protein lysates were prepared to determine FoxM1 and Sox2 changes 6 and 12 hours after irradiation. As loading controls, α-tubulin (cytoplasm) and TBP (nuclear) were used. Quantitation of these protein bands were shown in (D).</p

FoxM1 knockdown combined with <i>in vivo</i> irradiation significantly prolonged the survival of tumor bearing mice.

<p>(A) Kaplan-Meier survival curves of orthotopic tumor bearing mice. Control or FoxM1 expressing 387 GBM cells were intracranially injected into the brain of mice. Two weeks later tumor implantation, <i>in vivo</i> irradiation directed to the brains was performed (2 Gy daily does for 5 days). (B) Summary of survival and statistical significance. (C) Representative immunohistochemical images using FoxM1 and Sox2 antibodies on xenograft tumor sections. The brain sections were paraffin-embedded and stained with anti-FoxM1 and anti-Sox2 antibodies. H&E staining was used to visualize tissue histology.</p

FoxM1 expression levels in Clinical glioma specimens and association with patient survival.

<p>(A) Representative images of FoxM1 staining analysis using tissue microarray (TMA) sections. FoxM1 protein level of each glioma section was quantified according to the IRS method. (B) FoxM1 protein levels of each WHO glioma grade were compared using the IRS score (High = 4–12, Mild = 1–3, Low = 0). P-values were calculated using the Fisher’s exact test. (C and D) Kaplan-Meier survival curves and statistical analysis of glioma patients that were categorized based on the level of FoxM1. FoxM1 negative/low (blue) and high (red) groups were plotted. Overall survival (OS, left) and Progression-free survival (PFS, right) were compared by the Log rank test.</p