COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors
Yuanyuan Xie, … , Ping Chi, Yu Chen
Yuanyuan Xie, … , Ping Chi, Yu Chen
Published January 23, 2018
Citation Information: J Clin Invest. 2018;128(4):1442-1457. https://doi.org/10.1172/JCI94840.
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Research Article Oncology Therapeutics

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors

Abstract

Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling–dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.

Authors

Yuanyuan Xie, Zhen Cao, Elissa W.P. Wong, Youxin Guan, Wenfu Ma, Jenny Q. Zhang, Edward G. Walczak, Devan Murphy, Leili Ran, Inna Sirota, Shangqian Wang, Shipra Shukla, Dong Gao, Simon R.V. Knott, Kenneth Chang, Justin Leu, John Wongvipat, Cristina R. Antonescu, Gregory Hannon, Ping Chi, Yu Chen

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Figure 3

Genome-wide RNAi screen identifies key regulators of MAPK signaling–dependent ETV1 protein stability.

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Genome-wide RNAi screen identifies key regulators of MAPK signaling–depe...
(A) Schematic of the MAPK-ETV1 protein stability sensor construct with EGFP-ETV1 fusion protein and tdTomato expressed under the same promoter. (B) Immunoblots of EGFP and GAPDH in A2058 melanoma cells expressing EGFP or EGFP-flETV1, or EGFP-nETV1 and treated with DMSO (Veh), 1 μM vemurafenib, or MG132 for 8 hours. (C) FACS plot of tdTomato and EGFP fluorescence in A2058 cells expressing EGFP-nETV1-IRES-tdTomato treated with DMSO or 1 μM vemurafenib for 24 hours. (D) Histogram of the EGFP/tdTomato fluorescence ratio in EGFP-nETV1-IRES-tdTomato–expressing A2058 cells transduced with shRNA-miR against shSCR or BRAF (shBRAF) and treated with DMSO or 1 μM vemurafenib. (E) Schematic flow of screen. EGFP-nETV1-IRES-tdTomato–expressing A2058 cells were transduced with a genome-wide shRNA library. Cells were treated with vemurafenib for 24 hours prior to each sort. (F) Schematic of the role of selected hits in MAPK signaling–dependent regulation of ETV1 protein degradation. (G) Histogram of mCherry fluorescence of A375 cells expressing mCherry-nETV1 sensor treated with DMSO or 1 μM vemurafenib for 20 hours. (H) FACS plots of tGFP fluorescence linked to shRNA expression and mCherry-nETV1 fluorescence. A375 cells expressing mCherry-nETV1 and the indicated shRNA were transduced at a MOI of approximately 0.5 and treated with 1 μM vemurafenib or DMSO for an additional 20 hours.