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TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma

Seong, Bo Kyung A. and Dharia, Neekesh V. and Lin, Shan and Donovan, Katherine A. and Chong, Shasha and Robichaud, Amanda and Conway, Amy and Hamze, Amanda and Ross, Linda and Alexe, Gabriela and Adane, Biniam and Nabet, Behnam and Ferguson, Fleur M. and Stolte, Björn and Wang, Emily Jue and Sun, Jialin and Darzacq, Xavier and Piccioni, Federica and Gray, Nathanael S. and Fischer, Eric S. and Stegmaier, Kimberly (2021) TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma. Cancer Cell, 39 (9). pp. 1262-1278. ISSN 1535-6108. doi:10.1016/j.ccell.2021.07.003. https://resolver.caltech.edu/CaltechAUTHORS:20210902-234005496

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Abstract

Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.ccell.2021.07.003DOIArticle
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63473Related ItemRNA sequencing data
https://github.com/ndharia-broad/TRIM8_EwingRelated ItemCode
ORCID:
AuthorORCID
Seong, Bo Kyung A.0000-0002-1398-6230
Dharia, Neekesh V.0000-0001-5048-067X
Lin, Shan0000-0002-9699-0250
Donovan, Katherine A.0000-0002-8539-5106
Chong, Shasha0000-0002-5372-311X
Alexe, Gabriela0000-0002-5668-6297
Nabet, Behnam0000-0002-6738-4200
Ferguson, Fleur M.0000-0003-4091-7617
Darzacq, Xavier0000-0003-2537-8395
Piccioni, Federica0000-0003-1210-3210
Gray, Nathanael S.0000-0001-5354-7403
Fischer, Eric S.0000-0001-7337-6306
Stegmaier, Kimberly0000-0003-0218-7895
Additional Information:© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Received 3 August 2020, Revised 24 February 2021, Accepted 1 July 2021, Available online 29 July 2021. K.S. was supported by the National Cancer Institute R35 CA210030, R01 CA204915, and U54 CA231637, a St. Baldrick's Foundation Robert J. Arceci Innovation Award, and the Brian MacIsaac Sarcoma Foundation. B.K.A.S. was supported by Department of Defense PRCRP Horizon Award (CA181249), N.V.D. by the Julia's Legacy of Hope St. Baldrick's Foundation Fellowship, X.D. by U54 CA231641-01, and B.N. by the American Cancer Society Postdoctoral Fellowship PF-17-010-01-CDD. B.N. and N.S.G. were supported by the Katherine L. and Steven C. Pinard Research Fund and the Hale Center for Pancreatic Cancer. We thank Robert Tjian for scientific input on the super-resolution imaging experiments. Data and software availability. RNA-sequencing data generated in the manuscript is deposited in GEO: GSE63473. All codes used for the manuscript are deposited in github: https://github.com/ndharia-broad/TRIM8_Ewing. Author contributions. B.K.A.S. conceived the study, designed, and performed the experiments, analyzed the data, interpreted the results and wrote the manuscript. N.V.D. analyzed the CRISPR dependency screens, flow cytometry-based CRISPR screen, and RNA-seq data and interpreted the results. S.L. assisted with designing and performing the immunoprecipitation and the ubiquitination experiments, analyzed the data, and interpreted the results. K.A.D. performed mass spectrometry, analyzed the data, and interpreted the results. S.C. performed Airyscan confocal super-resolution imaging, analyzed the data, and interpreted the results. A.R. and A.C. performed in vivo studies, analyzed the data, and interpreted the results. L.R., G.A., B.A., and A.H. provided technical assistance, analyzed the data, and interpreted the results. B.N., F.M.F., and N.S.G. synthesized and provided the dTAGV-1 molecule. B.S. and E.J.W assisted with TRIM8 CRISPR knockout and EWS/FLI overexpression experiments, analyzed the data, and interpreted the results. J.S. provided technical assistance and intellectual input. F.P. assisted with designing the flow cytometry-based CRISPR screen, analyzed the data, and interpreted the results. E.S.F. analyzed the mass spectrometry data, interpreted the results, and supervised the study. K.S. conceived the study, designed the experiments, interpreted the results, and supervised and funded the study. All authors read, edited, and approved the final manuscript. Declaration of interests. K.S. has funding from Novartis Institutes for BioMedical Research, consults for and has stock options in Auron Therapeutics and served as an advisor for Kronos Bio and AstraZeneca all on topics unrelated to this manuscript. E.S.F. is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Jengu Therapeutics (board member), and Neomorph Inc., an equity holder in C4 Therapeutics, and is a consultant to Novartis, Sanofi, EcoR1 capital, Astellas, and Deerfield. The Fischer lab receives or has received research funding from Novartis, Ajax, Deerfield, and Astellas on topics unrelated to this manuscript. B.N. is an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148440, and WO/2018/148443). F.M.F., B.N., and N.S.G. are inventors on a patent related to the dTAG system and molecules described in this manuscript (WO/2020/146250). F.M.F. is a consultant to RA Capital and Santi Therapeutics. N.S.G. is a founder, SAB member and equity holder in Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member), and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, and Sanofi on topics unrelated to this manuscript. N.V.D. is a current employee of Genentech, Inc., a member of the Roche Group. K.A.D. is a consultant to Kronos Bio. Inclusion and diversity. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper received support from a program designed to increase minority representation in science.
Funders:
Funding AgencyGrant Number
NIHR35 CA210030
NIHR01 CA204915
NIHU54 CA231637
St. Baldrick's FoundationUNSPECIFIED
Brian MacIsaac Sarcoma FoundationUNSPECIFIED
Department of DefenseCA181249
NIHU54 CA231641-01
American Cancer SocietyPF-17-010-01-CDD
Katherine L. and Steven C. Pinard Research FundUNSPECIFIED
Hale Center for Pancreatic CancerUNSPECIFIED
Subject Keywords:fusion oncoproteins; EWS/FLI; Ewing sarcoma; TRIM8; E3 ligases; ubiquitination; protein degradation; neomorphic substrate; tumor dependency; oncogene overdose
Issue or Number:9
DOI:10.1016/j.ccell.2021.07.003
Record Number:CaltechAUTHORS:20210902-234005496
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210902-234005496
Official Citation:Bo Kyung A. Seong, Neekesh V. Dharia, Shan Lin, Katherine A. Donovan, Shasha Chong, Amanda Robichaud, Amy Conway, Amanda Hamze, Linda Ross, Gabriela Alexe, Biniam Adane, Behnam Nabet, Fleur M. Ferguson, Björn Stolte, Emily Jue Wang, Jialin Sun, Xavier Darzacq, Federica Piccioni, Nathanael S. Gray, Eric S. Fischer, Kimberly Stegmaier, TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma, Cancer Cell, Volume 39, Issue 9, 2021, Pages 1262-1278.e7, ISSN 1535-6108, https://doi.org/10.1016/j.ccell.2021.07.003.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:110716
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:07 Sep 2021 17:16
Last Modified:17 Sep 2021 22:45

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