The malignant trajectory from the notochord to chordoma and the role of the TBXT rs2305089 germline single nucleotide polymorphism

The rare bone cancer chordoma and its putative precursor the Benign Notochordal Cell Tumor (BNCT) express the notochordal transcription factor, TBXT. The aim of this study was to elucidate the role of rs2305089 germline single nucleotide polymorphism in TBXT in the pathogenesis of chordoma. A radiological and genotyping association study demonstrated that BNCTs are associated with chordomas (P<0.001) and with the rs2305089 polymorphism (P=0.002). We then engineered mesoderm/notochord models which differed only by their rs2305089 genotype. Expression of TBXT was higher and the gene regulatory network activated by TBXT was enriched in heterozygous compared to wild type cell lines. Heterozygous cell lines displayed enrichment of Wnt/beta-catenin and epithelial mesenchymal transition pathways while wild type cell lines were enriched for metabolic pathways and MTORC1 signalling. Moreover, the variant A allele induced faster cell migratory capacity together with changes in the expression of endoplasmic reticulum and intracellular transport mediators, thus suggesting that rs2305089 regulates notochord vacuoles as lysosome-related organelles in the aberrant regression of the notochord. In conclusion, we show for the first time that the rs2305089 variant allele is associated not only with chordoma but also with the BNCT and provide functional evidence that this SNP regulates notochordal cell differentiation in the pathogeneis of BNCTs and chordomas. Six cell lines which differed only by their genotype at the rs2305089 locus were engineered using CRISPR homology directed repair: three heterozygous (labelled 463N1-3) and three wild type/unedited cell lines (labelled 4610N1-3). Induced pluripotent stem cells were differentiated into mesoderm by 48 hours and mesenchymal stem cells (MSC) by 72 hours.