Abstract B50: Targeting EWS/FLI fusion oncoprotein stability/degradation in Ewing sarcoma
Bibliographic record
Abstract
Abstract Fusion-transcription factors (fusion-TFs) represent a class of oncoproteins that drive tumorigenesis by activating an aberrant transcriptional program to promote the development and survival of cancer cells. Unlike fusion oncoproteins involving readily druggable proteins such as kinases, fusion-TFs are difficult to therapeutically target. Recent studies have suggested protein degradation as a novel strategy in targeting oncogenic transcription factors. However, the molecular and functional mechanisms that regulate fusion-TFs’ degradation/stability are unknown. Ewing sarcoma is the second most common bone cancer in children and is driven by EWS-ETS fusion-TFs, most commonly EWS/FLI (~85%). EWS/FLI acts as a pioneer transcription factor to activate oncogenic gene expression by chromatin remodeling that drives the tumorigenesis and survival of Ewing sarcoma tumors. Despite this, there are no clinically validated targeted therapies against EWS/FLI. Ewing sarcoma has a relatively quiet genome and lacks recurrent mutations, which hinders target identification for therapeutic intervention. Thus, we examined whether a protein degradation strategy can be exploited to target EWS/FLI by investigating the molecular and functional mechanisms by which EWS/FLI protein stability/degradation is regulated. In a genome-scale, flow cytometry-based CRISPR-Cas9 screen, we discovered that tripartite motif containing 8 (TRIM8) is a novel E3 ubiquitin for EWS/FLI. Biochemical studies showed that TRIM directly binds and ubiquitinates EWS/FLI for proteasome-dependent degradation. Moreover, we identified TRIM8 as a top enriched and selective dependency in Ewing sarcoma in a genome-scale CRISPR-Cas9 depletion screen. We determined that TRIM8 knockout increased EWS/FLI protein levels and induced apoptosis of Ewing sarcoma cells in vitro and reduced tumor growth in vivo. Consistent with TRIM8 as an E3 ligase for EWS/FLI, overexpression of TRIM8 reduced EWS/FLI protein levels and decreased growth. TRIM8 lacking the E3 ligase domain (TRIM8E3) functioned as a dominant negative, leading to increased EWS/FLI and decreased growth of Ewing sarcoma cells. To investigate whether upregulated EWS/FLI protein expression is mediating the TRIM8 knockout phenotype, we engineered Ewing sarcoma cell line by concurrently knocking out endogenous EWS/FLI using CRISPR and expressing degradable EWS/FLI (FKBP12F36V-EWS/FLI) using the dTAG system. The dTAG molecule is a heterobifunctional molecule that specifically binds to FKBP12F36V and an E3 ligase complex for targeted protein degradation. Using this degradable EWS/FLI cell model, we showed that TRIM8 suppression phenotype can be rescued by degrading the upregulated EWS/FLI protein expression to control levels. Our results demonstrate that the protein stability of fusion-TF oncoproteins is highly regulated to maintain the expression at a precise level and highlight the critical importance of oncogene dosage in cancer cell survival. Citation Format: Bo Kyung Alex Seong, Shan Lin, Katherine Donovan, Amanda Robichaud, Bjorn Stolte, Emily Wang, Neekesh Dharia, Behnam Nabet, Federica Piccioni, Nathanael Gray, Eric Fischer, Kimberly Stegmaier. Targeting EWS/FLI fusion oncoprotein stability/degradation in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B50.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".