YBX1-interacting small RNAs and RUNX2 can be blocked in primary bone cancer using CADD522

Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity that produces novel morphological features plus clinical behaviour serendipitously makes an excellent model to study metastasis. Here we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) act as a constitutive tumour suppressor mechanism by blunting potential metastatic protein-RNA interaction, a mechanism lost in PBC progression. We demonstrate a gradual loss of tRNAGlyTCC cleavage into 5' end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade clinical samples. Contrarily, we show recurrent activation of microRNA-140 leading to upregulated RUNX2 in high-grade clinical samples. Mechanistically, loss and gain of function studies using antisense LNA inhibitors plus RNA mimics revealed that ectopic tRF-GlyTCC reduced RUNX2 and dispersed tumour sphere architecture in vitro. This mode of RNA silencing is sequence specific because tRF-GlyTCC and RUNX2 share a motif in their 3' ends. Rather than perform in an AGO2-dependent complex to achieve gene silencing, this motif matches the YBX1 recognition site known to stabilise metastatic messenger RNAs, which iCLIP experiments revealed includes RUNX2. Discovery of this novel tRF-GlyTCC-RUNX2 axis led to in vivo development of the RUNX2 transcription factor/ATP synthase inhibitor CADD522 as a PBC treatment in mice. Overall design: Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP-seq) of YBX1 protein done in triplicate