The interaction between RUNX2 and core binding factor beta as a potential therapeutic target in canine osteosarcoma

Abstract

Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFβ) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti-tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFβ inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line-dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFβ and alterations in expression of RUNX2 target genes. We also show that addition of CBFβ inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti-proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFβ, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti-tumour activities we describe here.

Keywords: RUNX2; canine sarcoma; core-binding factor beta; novel therapeutic targets; osteosarcoma; transcription factor.

Figure 1.. Cell proliferation and clonogenic growth is suppressed by small molecule inhibitors of CBFβ/RUNX2 binding in canine OSA cell lines.

Resazurin assay was used to assess cellular proliferation/viability in (A) cells treated with a range of concentrations (0-100 μM) of AI-10-104, AI-14-91 or AI-4-88 (negative control compound), or vehicle control for 48 hours, and (B) cells treated with 20 μM of AI-10-104, AI-14-91 or AI-4-88, or vehicle control after 24, 48, or 72 hours-treatment. (C) Representative images from clonogenic assays with respective surviving fractions. Data (mean±SEM) reported as percentage of control and analyzed by one-way or two-way ANOVA followed by a Bonferroni test (**p <0.01, ***p <0.001, ****p <0.0001).

Figure 2.. Inhibitors of CBFβ reduce CBFβ/RUNX2 binding in canine OSA cell lines.

(A) Representative western blot images of CBFβ (22 kDa) and RUNX2 (55 kDa) in untreated Abrams, HMPOS, D-17 and Grade cells. (B) Representative images of the immunoblotting analysis and histograms expressing quantification of CBFβ after immunoprecipitation of RUNX2 in protein lysates from HMPOS and D-17 cells treated with AI-10-104, AI-14-91 or AI-4-88 at 20 μM, or vehicle control for 6 hours.

Figure 3.. Necrotic and apoptotic cell death was triggered in canine OSA cell lines treated with small molecule inhibitors of CBFβ/RUNX2 binding.

Canine OSA cells treated with AI-10-104, AI-14-91 or AI-4-88 at 20 μM, or vehicle control for 48 hours were employed to evaluate the cell-death by means of (A) a caspase-3/-7 activity assay and (B) a bivariate PI/Annexin-V analysis. Data (mean±SEM) were calculated as percentage of control (untreated cells) and analyzed by two-way ANOVA multiple test followed by a Bonferroni test (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).

Figure 4.. CBFβ inhibitors disrupted the cell cycle in canine OSA cells.

Flow cytometry histograms (left) and quantitation of cell cycle distribution (right) of (A) Abrams and (B) D-17 cells stained with PI after treatment with vehicle, or 20 μM AI-10-104, AI-14-91 or AI-4-88 (negative control compound) for 24 hours. Data (mean±SEM) were calculated as percentage of control (untreated cells) and analyzed by two-way ANOVA multiple test followed by a Bonferroni test (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).

Figure 5.. Migration and invasive capabilities were significantly inhibited in canine OSA cells by CBFβ inhibitors.

D-17 and HMPOS cells plated on upper matrigel-coated or control inserts were treated with 20 μM AI-10-104 or AI-4-88 (negative control compound), or vehicle and subjected to transwell invasion/migration assays for 20 hours. (A) Representative phase-contrast microscopy images (20X) of transwell invasion and migration assays. (B) Histograms expressing percentage of migrating and invading cells after 20 hours of incubation. (C) Invasion index was calculated by dividing percentage invasion of treated cells by the percentage invasion of vehicle cells. Data (mean±SEM) analyzed by one-way or two-way ANOVA followed by a Bonferroni test (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).

Figure 6.. Small molecule inhibitors of CBFβ/RUNX2 binding modulated gene expression of RUNX2 targets in canine OSA cell lines.

Relative mRNA expression levels of VEGFA, Cdkn1a and Bglap were analyzed by qRT-PCR and normalized versus geometric mean of housekeeping genes GAPDH, Rps5, and Hnrnph1. Data (mean±SEM) were analyzed by two-way ANOVA multiple test followed by a Bonferroni test (*p <0.05, **p <0.01, ***p <0.001).