UC Davis

Osteosarcoma (OS) is the most common primary bone malignancy in humans and canines, and in humans primarily affects younger patients 10-14 years of age. While considerable efforts have been put forth in new therapeutic approaches to this disease, the treatment and prognosis for OS has changed very little since the 1980s. Targeted therapeutics have made considerable progress in other cancer types, leveraging characteristics of cancer cells which differentiate them from that of normal healthy cells. In comparison to other cancers, OS is highly heterogeneic, and no single unifying driver mutation has yet been found. Development of a therapy which could overcome the high degree of heterogeneity amongst OS tumors could go a long way in improving the lives of OS patients. Targeting protein translation has been proposed as one mechanism by which to overcome tumor heterogeneity, and this dissertation focuses on studying a noncanonical role of core binding factor beta (CBFβ) as a regulator of protein translation, and elucidating whether this could represent a potential therapeutic target for OS. Utilizing a wide array of in vitro assays, we have been able to demonstrate that loss of CBFβ reduces protein expression of RUNX2 in a post-transcriptional manner, and this decrease in RUNX2 protein level is not fully explained by alterations in RUNX2 stability brought about by loss of its binding partner CBFβ. Additionally, we demonstrate that loss of CBFβ also causes a decrease in global protein translation, and confirmed an interaction between CBFβ and hnRNPK which has thus far only been observed in breast cancer cells. Importantly, this interaction with hnRNPK is said to be the mechanism by which CBFβ influences protein translation, and our results corroborate those observed in breast cancer cells and suggest CBFβ may also perform this role in OS. Reports of the interactions between CBFβ and hnRNPK or RUNX2 allude to mutual exclusivity in interaction, and with the transcriptional role of CBFβ accomplished via binding to RUNX proteins, and the translational role of CBFβ accomplished via binding to hnRNPK, it is entirely possible these two roles are antagonistic in some fashion. To investigate the relevance of certain CBFβ residues in terms of this translational role of CBFβ, and avoid confounding variables from the transcriptional role of CBFβ, we utilized point mutations to interrupt CBFβ-RUNX2 interaction. Using various in vitro assays, we validated key residues of CBFβ which are involved in its interaction with RUNX2, re-introduced this mutant form into CBFβ knockout cells, and measured alterations to RUNX2 interaction and nuclear shuttling. We confirmed that our mutant displays reduced binding to RUNX2, and drastically reduced nuclear shuttling. Lastly, we expanded our studies from RUNX2 to the entire genome and proteome. Encouraging data thus far had suggested CBFβ may play a role in protein translation, and necessary next steps were to assess which proteins CBFβ may be interacting with in performance of this role, and elucidate which proteins may be under the translational purview of CBFβ. Using immunoprecipitation mass spectrometry we identified numerous specific interactors of CBFβ, with high enrichment in pathway analysis terms associated with protein translation. Additionally, using two different methods we generated a list of proteins which may be under the translational purview of CBFβ, and found strong enrichment of numerous cancer-associated terms among this list. These studies establish that CBFβ participates in protein translation in OS, with many genes under its purview associated strongly with cancer in general, and OS specifically. This provides justification for future studies delving deeper into this novel role of CBFβ, and opens up another mechanism by which protein translation could be targeted therapeutically in OS.