Inhibition of Core Binding Factor Transcriptional Complex Provides a Novel Pharmacological Target for Osteosarcoma
Authors: Godinez Dayn R. , Oldberg Nicholas , Wittenburg Luke A. 
Published: May 2022

Osteosarcoma (OS) is the most common primary malignant bone tumor in canines and humans. However, there have been no new successful therapies that improve long‐term survival in several decades. This indicates the need for novel therapeutic targets for treating OS. Core binding factor is a heterodimeric transcriptional complex, comprised of proteins runt‐related transcription factor 2 (RUNX2) and core‐binding factor subunit Beta (CBFβ), that promotes transcription of genes related to bone formation, osteoblast differentiation, and bone mineralization. RUNX2 protein expression is deregulated in OS tumors which contributes to chemoresistance, increased proliferation, and defective differentiation, suggesting it could be a therapeutic target in OS. This study utilizes small molecule allosteric inhibitor of CBFβ, AI‐14‐91, to target the interaction of RUNX2 and CBFβ, thereby reducing RUNX2 binding to target genes, in order to evaluate the CBFβ and RUNX2 interaction as a pharmacological target in OS. We hypothesized that AI‐14‐91 will reduce the malignant phenotype of human OS cell lines by disrupting the RUNX2‐CBFβ interaction.

In vitro assays with compound AI‐14‐91 were performed with a panel of unique human OS cell lines (LM7, MG‐63, Saos‐2, SJSA‐1, and U2OS). This panel contains unique genetic mutations in key tumor‐related genes such as p53, MDM2, Rb1, or CDKN2A. RUNX2 knockout (KO) and CBFβ KO cell lines were also generated from the parental U2OS by CRISPR/Cas9 editing, which was confirmed via sequencing and western blot. Concentration‐ and time‐dependent proliferation assays were performed with all cell lines, demonstrating AI‐14‐91 IC50 values ranging from 13‐25 µM across the panel. Clonogenic assays showed AI‐14‐91 treatment significantly reduced colony formation in the majority of cell lines. Flow cytometry revealed cell cycle progression was altered in the parental U2OS and the RUNX2 KO and CBFβ KO cell lines with a significant decrease of cell number in G0/G1 phase and an increase in S phase upon 48 exposure to 20 µM AI‐14‐91. Apoptotic and necrotic cell populations in parental U2OS and KO lines were quantified by flow cytometry bivariate analysis with Annexin V/PI co‐stain. Batch 3’ Tag‐Seq RNAseq analysis was performed on parental U2OS cells exposed to 5 or 20 µM AI‐14‐91 for 6 or 48 hours and on RUNX2 KO and CBFβ KO cell lines. Differential gene expression (DGE) and enrichment analysis was performed to evaluate pertinent transcriptional pathways altered by inhibitor treatment, and to compare the effects of inhibitor treatment with knockout of putative inhibitor targets.

Treatment with AI‐14‐91 has shown desirable antitumor effects in human OS cell lines that may be independent of the absence of proteins RUNX2 or CBFβ. Further analysis into DGE pathways combined with ChIP assays will help delineate mechanisms of action and identify additional potential targets for combination treatments. Orthotopic mouse models utilizing the metastasizing LM7 cell line with provide further information on the potential clinical utility of targeting the RUNX2‐CBFβ interaction in OS.