Peer Reviewed Cancer
Posted July 27, 2021
Dr. Bo Kyung Seong, Ph.D., Dana-Farber Cancer Center
Ewing sarcoma (EWS) is the second most common bone cancer that affects children and adolescents.1 Treatment options, which usually include chemotherapy, radiation, and surgery when possible, have not changed in more than a decade. The oncogene EWS/FLI is responsible for the development and survival of many Ewing sarcoma tumors; but, because targeted therapies for oncogenes often rely on small-molecule inhibitors and the transcription factors have been difficult to target using this approach, targeted therapy for EWS has not yet been successfully developed.
With the help of Dr. Kimberly Stegmaier at Dana-Farber Cancer Institute, Dr. Bo Kyung Seong focuses on the integration of “omic” approaches and Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) gene editing technology to screen for and identify genes that can regulate protein stability. The lab’s previous work developed a flow cytometry-based CRISPR-Cas9 screening, which identified TRIM8, an E3 ubiquitin ligase, as a potential regulator of EWS/FLI protein stability. With funding from a Fiscal Year 2018 Peer Reviewed Cancer Research Program (PRCRP) Horizon Award, Dr. Seong hypothesized that TRIM8 is a novel E3 ubiquitin ligase that tags EWS/FLI proteins for degradation, and that TRIM8 knockout increases EWS/FLI protein expression levels to a point which EWS cells cannot tolerate, resulting in decreased cell growth. Based on his hypothesis, Dr. Seong aimed (1) to determine if TRIM8 regulates EWS/FLI protein expression in EWS cells; and (2) to analyze whether the functional phenotypes of TRIM8 knockout are controlled and changed by elevated protein levels of EWS/FLI. While the work supported by the PRCRP is ongoing, Dr.Seong has made significant progress in characterizing TRIM8 function in EWS cells.
Dr. Seong began by confirming a relationship between TRIM8 and EWS/FLI. Using TRIM8 targeting sgRNAs to knockout TRIM8 expression, Dr. Seong observed that in the absence of TRIM8, EWS/FLI expression increased in EWS cells. Moreover, Dr.Seong observed that overexpression of TRIM8 resulted in decreased EWS/FLI expression, supporting his hypothesis that TRIM8 is an E3 ligase for EWS/FLI. He then generated a TRIM8 mutant (TRIM8ΔRING) that would knock out the molecule’s function as a ligase. Overexpression of TRIM8ΔRING not only failed to downregulate EWS/FLI expression, Dr. Seong actually observed an increase in EWS/FLI expression. Overexpression of TRIM8 in the presence of a proteasome inhibitor, a molecule that blocks the cellular complexes that break down proteins, rescued TRIM8-mediated degradation of EWS/FLI. Taken together, these findings suggest that TRIM8-mediated regulation of EWS/FLI occurs at the post-translational level.
Dr. Seong was also able to test whether TRIM8 could directly bind and ubiquitinate (tag for degradation) EWS/FLI. By performing co-immunoprecipitation and ubiquitination assays with TRIM8 and EWS/FLI over-expression, Dr. Seong found that TRIM8 does directly interact with EWS/FLI. In addition, TRIM8 expression leads to polyubiquitination of EWS/FLI, which subsequently leads to protein degradation.
In the first year of the award, Dr. Seong and his team were able to confirm that TRIM8 does directly interact with EWS/FLI. Further research supported by the PRCRP Horizon Award will focus on the second project aim to explore the functionality of TRIM8 in relation to EWS/FLI. Dr. Seong has presented these findings at several scientific conferences, including a pediatric-focused conference named “Advances in Pediatric Cancer Research,” which is organized by the American Association for Cancer Research. Using his findings to date, Dr. Seong will conduct additional studies in order to validate the TRIM8 dependency in EWS cell lines and to clarify the molecular mechanisms surrounding these two proteins. By improving the research community’s understanding of TRIM8 and EWS/FLI, Dr. Seong may be paving the way for new targeted therapies for patients with Ewing sarcoma.
Public and Technical Abstracts: Targeting Pediatric Fusion Oncoproteins via Protein Degradation
Last updated Thursday, May 26, 2022