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. 2023 Mar 5;39:100474.
doi: 10.1016/j.jbo.2023.100474. eCollection 2023 Apr.

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

Darrell Green  1 , Archana Singh  2 , Victoria L Tippett  3 , Luke Tattersall  3 , Karan M Shah  3 , Chileleko Siachisumo  4 , Nicole J Ward  2 , Paul Thomas  2   5 , Simon Carter  6 , Lee Jeys  6 , Vaiyapuri Sumathi  7 , Iain McNamara  8 , David J Elliott  4 , Alison Gartland  3 , Tamas Dalmay  2 , William D Fraser  1   9
Affiliations
Free PMC article

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

Darrell Green et al. J Bone Oncol. .
Free PMC article

Abstract

Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity produces novel morphological features and clinical behaviour that serendipitously makes PBC an excellent metastasis model. Here, we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) perform as a constitutive tumour suppressor mechanism by blunting a potential pro-metastatic protein-RNA interaction. This mechanism is reduced in PBC progression with a gradual loss of tRNAGlyTCC cleavage into 5' end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade patient tumours. We detected recurrent activation of miR-140 leading to upregulated RUNX2 expression in high-grade patient tumours. Both tRF-GlyTCC and RUNX2 share a sequence motif in their 3' ends that matches the YBX1 recognition site known to stabilise pro-metastatic mRNAs. Investigating some aspects of this interaction network, gain- and loss-of-function experiments using small RNA mimics and antisense LNAs, respectively, showed that ectopic tRF-GlyTCC reduced RUNX2 expression and dispersed 3D micromass architecture in vitro. iCLIP sequencing revealed YBX1 physical binding to the 3' UTR of RUNX2. The interaction between YBX1, tRF-GlyTCC and RUNX2 led to the development of the RUNX2 inhibitor CADD522 as a PBC treatment. CADD522 assessment in vitro revealed significant effects on PBC cell behaviour. In xenograft mouse models, CADD522 as a single agent without surgery significantly reduced tumour volume, increased overall and metastasis-free survival and reduced cancer-induced bone disease. Our results provide insight into PBC molecular abnormalities that have led to the identification of new targets and a new therapeutic.

Keywords: CADD522; CADD522, computer aided drug design molecule 522; CI, confidence interval; CNV, copy number variant; CS, chondrosarcoma; CTC, circulating tumour cell; DE, differentially expressed; ES, Ewing sarcoma; HD, high definition; HR, hazard ratio; OS, osteosarcoma; RBP, RNA binding protein; RNU6-1, U6 small nuclear 1; ROI, region-of-interest; Rnl, T4 RNA ligase; SNV, single nucleotide variant; SV, structural variant; bone cancer; iCLIP, individual nucleotide resolution cross-linking and immunoprecipitation; mRNA, messenger RNA; miRNA; miRNA, microRNA; piRNA, piwi interacting RNA; sRNA, small RNA; small RNA; tRF; tRF, transfer RNA fragment; tRNA, transfer RNA; ysRNA, Y RNA-derived sRNA.

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
sRNA-seq schematic, 8% PAGE and heat map-based normalised sRNA expression hierarchical linear scale. A. Traditional sRNA-seq employs Rnl2 to ligate a 3′ DNA adapter and Rnl1 to ligate a 5′ RNA adapter to an sRNA sample. The product is then converted to cDNA before undergoing next generation sequencing. B. Commercial adapters are a fixed sequence. Rnl2 and Rnl1 are biased for ligating adapters and sRNAs where there is a good complementarity between the sequences or annealing efficiency. sRNAs that do not have a good annealing efficiency are not ligated to adapters and are not sequenced. HD adapters contain four random nucleotides on the ligating ends of commercial adapters creating a 28 sequence pool that increases the annealing efficiency between sRNAs and adapters. C. 8% PAGE as part of the size selection step. Upper band (blue arrow) revealed tRF and ysRNA libraries. Middle band (black arrow) were miRNAs. Adapter dimers (red arrow) were omitted to increase depth for tRF and miRNA sequencing. D. Statistically significant tRF expression comparisons between control, low-grade, intermediate-grade and high-grade clinical CS samples. E. Statistically significant miRNA expression comparisons between control, low-grade, intermediate-grade and high-grade clinical CS samples. Z-score refers to high (red) and low (blue) sRNA expression using normalised values when compared with the mean of total sequencing reads.
Fig. 2
Fig. 2
Mir-140, mir-320a, mir-486 and RUNX2 expression is associated with lower sarcoma patient overall survival. The relationship between selected DE transcripts and sarcoma patient overall survival was assessed by Kaplan–Meier plotter (https://kmplot.com/analysis), which includes gene (mRNA, miRNA) and protein expression and survival data in over 30,000 samples from 21 tumour types. Samples were divided into two groups with high (red) and low (black) expression. Hazard ratios (HR) and logrank p values are shown. miR-140 expression had a lower prognosis (HR, 1.34) but this was non-significant.
Fig. 3
Fig. 3
tRF-GlyTCC loss-of-function is associated with CS progression. A. Full length tRNA secondary structures were generated using tRNAscan-SE and Forna . tRNAGlyTCC with a 5′ end cleavage between C and U. tRNALysTTT with a 5′ end cleavage between U and U. tRNAAsnGTT with a 3′ end cleavage between A and G. B. Normalised count read matrix represented as a box and whisker plot for tRF-GlyTCC expression between tissue/tumour grades. C. Northern blot for miR-140 using an antisense 3′ end probe in control, low-grade, intermediate-grade and high-grade clinical samples. RNU6-1 and total RNA show equal (but low) loading to the membrane. D. CS micromass’ stained with DAPI to show nuclear DNA and Texas Red-X Phalloidin to show F-actin. Photomicrographs were taken at the most abundant cellular region of each micromass. Scale bars are 100 µm. E. Sequence logo analysis for tRF-GlyTCC, YBX1 and RUNX2. F. Using stored RNA from sequencing experiments and RNA isolated from micromass experiments, ectopic tRF-GlyTCC reduced RUNX2 mRNA and increased G. YBX1 mRNA. Data was normalised to ACTB expression. Statistical significance was calculated using an unpaired t-test.
Fig. 4
Fig. 4
YBX1 physically interacts with RUNX2 3′ UTR. A. PAGE gel showing YBX1 pull down. B. mRNA-seq reveals mRNA regions bound to by YBX1. C. YBX1 physically interacts with several regions across the RUNX2 mRNA with marked enrichment in the 3′ UTR.
Fig. 5
Fig. 5
CADD522 significantly increases OS metastasis-free survival and ES overall survival. A. C15H13Cl2NO3 chemical structure. B. CS, OS and ES cell (GFP-luciferase tagged for the animal experiments) viability and proliferation in response to CADD522 in vitro after 72 h. ***p < 0.005, **p < 0.01. Each dot is one replicate. Four replicates were tested in three independent experiments. C. OS and ES mineralisation measured over 7 d treated with a CADD522 dose range. Data shown is mean +/- SD from three independent experiments, four wells per experiment. Plot shows a one-way ANOVA on the mean of the independent experiments with Dunnett’s multiple comparison (n = 3). ****p < 0.001, **p < 0.01. D. Tumour luminescence in CADD522 treated OS animals. Luminescence decreases (21 d) after a spike (14 d) in the control mice because OS tumours are centrally necrotic, but the tumour volume still increases. E. Tumour luminescence in CADD522 treated ES animals. F. Tumour volumes in OS animals when treated with CADD522 versus non-treated controls. G. Tumour volumes in ES animals when treated with CADD522 versus non-treated controls. H. Metastasis-free survival in OS animals when treated with CADD522 versus non-treated controls. I. Overall survival in ES animals when treated with CADD522 versus non-treated controls. No metastases were detected in the ES model.
Fig. 6
Fig. 6
CADD522 reduces PBC-induced bone disease. A. OS micro-CT image showing PBC-induced bone disease (severe mixed effects of ectopic bone formation and lytic bone destruction). B. Bone volume quantification and comparison to the non-tumour bearing contralateral (CL) leg. *p < 0.05. C. ES micro-CT image showing a similar architectural phenotype to OS. D. Bone volume quantification and comparison to the non-tumour bearing contralateral (CL) leg. E. Representative OS tumour H&E sections. F. Representative ES tumour H&E sections. Scale bars are 50 µm.
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