Abstract A145: CBFβ supports global protein translation in osteosarcoma and may provide a new therapeutic target
Authors: Oldberg Nicholas A , Godinez Dayn R , Wittenburg Luke A 
Published: December 2023

Background: Core binding factor beta (CBFβ) is 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 affinity for DNA. RUNX2 is the master regulator of bone growth and differentiation, and its binding to CBFβ is of particular interest to osteosarcoma (OS) research. Notably, increased expression of each is correlated with poor disease-free and overall survival in numerous sarcomas. CBFβ’s transcriptional role is well-understood but recent research in breast cancer cells proposes a non-canonical role in the regulation of protein translation, influencing translation of hundreds of mRNA transcripts via binding with heterogenous nuclear ribonucleoprotein K (hnRNPK). The binding of CBFβ to hnRNPK or RUNX1 is reportedly mutually exclusive, suggesting possible interplay between the two roles. We hypothesize that CBFβ regulates protein translation in OS, participating in the translation of RUNX2 and other oncoproteins, promoting a malignant phenotype. Furthermore, we propose that disrupting its translational role may result in an antitumor effect. While CBFβ’s transcriptional activities are well-characterized, we endeavored to isolate its translational role and investigate its contribution to OS.

Methods: CRISPR/Cas9 was used to generate a CBFβ knockout (KO) U2OS cell line, while site-directed mutagenesis of CBFβ and lentiviral transduction of U2OS CBFβ KO cells was used to generate cell lines expressing FLAG-tagged wild type (WT) mutant CBFβ. Western Blotting (WB) was used to assess changes in protein expression and subcellular localization while RUNX2 mRNA expression was measured via qPCR and RNAseq. CBFβ’s interacting partners were identified by Immunoprecipitation Mass Spectrometry (IP-MS). Cellular electro-thermal shift assay was used to evaluate influences of CBFβ mutations on RUNX2 and hnRNPK binding. The Click-IT system followed by WB and IP-MS were used to assess global protein translation and synthesis rates of specific proteins, respectively.

Results: CBFβ and hnRNPK interact in U2OS cells and loss of CBFβ leads to a decrease in global protein translation. This results in decreased RUNX2 protein, but not mRNA, expression. Re-introduction of WT CBFβ caused RUNX2 protein levels to recover while expression of CBFβ with key mutations in the binding interface abrogated this effect. Importantly, we observed this was not due to direct protection of RUNX2 from proteasomal degradation by WT CBFβ. We identified a cohort of proteins that appear to be translationally regulated by CBFβ and investigated the impact of CBFβ mutations on its translational activities and RUNX2 binding. We also identified a list of putative CBFβ protein-protein interactions in human OS cells that are altered by these mutations.

Conclusions: CBFβ appears to participate in protein translation in OS, regulating the expression of numerous oncoproteins including RUNX2. Key mutations in CBFβ designed to interrupt RUNX2 binding also affect this non-canonical activity of CBFβ.

Citation Format: Nicholas A Oldberg, Dayn R Godinez, Luke A Wittenburg. CBFβ supports global protein translation in osteosarcoma and may provide a new therapeutic target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A145.