Mechanisms of Toxicity Induced by EWS/FLI1 Overdose in Ewing Sarcoma

项目总结/文摘

PROJECT SUMMARY/ABSTRACT EWS fusion oncoproteins are critical drivers in a number of malignancies, including the pediatric solid tumor Ewing sarcoma, the second most common pediatric cancer involving bone. In the case of Ewing sarcoma, the fusion of EWS to the ETS transcription factor FLI1 generates an oncogenic pioneer transcription factor that alters a distinctive set of regulatory elements to control oncogenic gene expression. The impact of repression of the EWS/FLI1 fusion with approaches such as shRNA, CRISPR, or targeted protein degradation is now well described and known to be deleterious to Ewing sarcoma cells. In contrast, the potential of EWS/FLI1 overdose is just newly described and has been understudied. We recently discovered a powerful and novel selective dependency in Ewing sarcoma on TRIM8, an E3 ubiquitin ligase. Remarkably, TRIM8 controls the levels of EWS/FLI1 protein, and its inactivation leads to an increase in EWS/FLI1 protein levels that is not tolerated by tumor cells. Ewing sarcoma cells must thus carefully regulate the levels of EWS/FLI1 within a narrow “just right,” “Goldilocks” range. Either too little or too much fusion protein is lethal. The mechanisms that control optimal levels of EWS/FLI1 and that mediate cell lethality from high levels of the fusion protein are unknown. We expect that defining these mechanisms will reveal important principles of Ewing sarcoma biology that may also be applicable to other EWS fusion proteins, and that this knowledge may point to new therapeutic strategies. The overall goal of this proposal is thus to determine the mechanisms of EWS/FLI1 induced toxicity following TRIM8 loss in Ewing sarcoma through three aims. The gene regulation properties of EWS/FLI1 are highly dependent on the formation of biomolecular condensates, which in turn require the phase transition properties of the EWS disordered prion-like domain. Similarly, TRIM8 has been described to have a role in condensate formation. Thus, in Aim 1, we will determine the relationship between TRIM8 and EWS/FLI1 biomolecular condensates with the hypothesis that that EWS/FLI1 regulation by TRIM8 is dependent on condensate formation and that, in turn, loss of TRIM8 leads to changes in condensates. In Aim 2, we will determine the impact of TRIM8 loss on epigenetic states in Ewing sarcoma including 3D interactions. In Aim 3, we will identify the mechanisms by which EWS/FLI1 overdose is toxic. Based on gene expression and ORF screening data, we will lead this aim with the hypothesis that the glutamate receptor GRM2 mediates toxicity downstream of EWS/FLI1 overdose. New therapeutic approaches distinct from traditional cytotoxic chemotherapy and local control measures are needed for patients with Ewing sarcoma as little progress has been made in treating high-risk patients over several decades. These proposed studies exploring an innovative strategy of oncogene overdose have potential for future translational impact in addition to revealing basic underlying mechanisms of disease.