Decoupling the Transcriptional and Translational Roles of Core Binding Factor Beta to Identify Novel Therapeutic Targets in Osteosarcoma
Authors: Oldberg Nicholas A. , Godinez Dayn R. , Wittenburg Luke A. 
Published: May 2022

Core binding factor beta (CBFβ) functions as a binding partner to the RUNX family of DNA binding transcription factors (RUNX1‐3) and acts as a transcriptional co‐activator by allosterically increasing their DNA binding. According to The Cancer Genome Atlas Program (TCGA) and Genotype‐Tissue Expression (GTEx) datasets, high CBFβ expression is correlated with poor disease‐free and overall survival across 17 cancer types, including sarcomas. Of particular interest to osteosarcoma (OS) research is the interaction of CBFβ with RUNX2, the master regulator of bone growth and differentiation, also dysregulated in aggressive OS and implicated in chemoresistance. Recent research supports a non‐canonical role of CBFβ as a regulator of protein translation initiation. In breast cancer cells, this translation‐associated activity occurs via interactions with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and allows CBFβ to influence translation of hundreds of mRNA transcripts. Binding of CBFβ to hnRNPK or RUNX1 appears mutually exclusive, suggesting possible competition between these two roles.

We hypothesized that CBFβ plays a role in the translation of RUNX2 and RUNX2‐target gene mRNAs in OS and that disruption of this role may result in an antitumor effect. The intertwined nature of the two roles of CBFβ hampers study of each in OS. Thus, we aimed to decouple them to identify which presents the most viable therapeutic target in OS.

Using CRISPR/Cas9 we generated a CBFβ knockout (KO) U2OS cell line and evaluated RUNX2 protein and mRNA levels via western blot (WB) and qRT‐PCR, respectively. Cycloheximide chase assay and proteasome inhibition were used to evaluate RUNX2 stability and degradation, respectively. Interactions between CBFβ and RUNX2 or hnRNPK were assessed via co‐immunoprecipitation. Site‐directed mutagenesis (SDM) of CBFβ and transfection of U2OS CBFβ KO cells with FLAG tagged CBFβ mutants was used to prevent RUNX2‐CBFβ interaction. WB was used to evaluate changes in subcellular CBFβ localization. Global, as well as protein‐specific, changes in de novo protein synthesis were evaluated using the Click‐IT system. Finally, in silico modeling was used to design a peptide that mimics a 10 amino acid sequence on RUNX2 in the CBFβ binding interface, which was then evaluated in vitro using a recombinant RUNX2‐CBFβ pulldown assay.

Our results demonstrate that loss of CBFβ leads to reduced RUNX2 protein expression without changes in RUNX2 mRNA levels. RUNX2 protein half‐life is not reduced in CBFβ nor does proteasome inhibition rescue RUNX2 protein levels. CBFβ interacts with hnRNPK in OS cells, and through this binding may perform a role in oncoprotein translation in OS, specifically modulating protein levels of RUNX2. SDM of CBFβ identified important residues for RUNX2 binding, which when targeted via a peptide resulted in a nearly 50% reduction in CBFβ and RUNX2 interaction. Ribosome footprinting combined with RNAseq and immunoprecipitation‐mass spectrometry utilizing WT U2OS, CBFβ mutants and peptide‐inhibited cells will provide further information on the translational role of CBFβ and potential therapeutic targets in OS.