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Dissolution of oncofusion transcription factor condensates for cancer therapy

Nature Chemical Biology volume 19, pages 1223–1234 (2023)Cite this article

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Abstract

Cancer-associated chromosomal rearrangements can result in the expression of numerous pathogenic fusion proteins. The mechanisms by which fusion proteins contribute to oncogenesis are largely unknown, and effective therapies for fusion-associated cancers are lacking. Here we comprehensively scrutinized fusion proteins found in various cancers. We found that many fusion proteins are composed of phase separation-prone domains (PSs) and DNA-binding domains (DBDs), and these fusions have strong correlations with aberrant gene expression patterns. Furthermore, we established a high-throughput screening method, named DropScan, to screen drugs capable of modulating aberrant condensates. One of the drugs identified via DropScan, LY2835219, effectively dissolved condensates in reporter cell lines expressing Ewing sarcoma fusions and partially rescued the abnormal expression of target genes. Our results indicate that aberrant phase separation is likely a common mechanism for these PS–DBD fusion-related cancers and suggest that modulating aberrant phase separation is a potential route to treat these diseases.

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Fig. 1: Identification of cancer-associated PS–DBD fusion proteins as candidates for aberrant phase separation.
Fig. 2: The FUS–ERG fusion protein drives phase separation in cellulo and in vitro.
Fig. 3: DBD domains tend to fuse PS-related regions, and such fusions are likely to drive downstream transcription.
Fig. 4: DropScan, a high-content screening method for identifying phase separation-modulating compounds.
Fig. 5: LY2835219 dissolves condensates via activation of lysosomes.
Fig. 6: LY2835219 reduces droplet numbers and rescues abnormal gene expression.

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Data availability

All sequencing data that support the findings of this study have been deposited in the National Center for Biotechnology Information GEO and are accessible through the GEO Series accession numbers GSE194374 and GSE232072. Drug screening data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

All custom scripts have been made available at https://github.com/TingtingLiGroup/DropScan.

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Acknowledgements

We acknowledge L. Yu and Y. Lin (Tsinghua University) and Z. Qi (Peking University) for providing plasmids as gifts. We are grateful to the Center of Pharmaceutical Technology (Tsinghua University) for high-content screening support. We are also grateful to the Nikon Biological Imaging Center (Tsinghua University) for confocal imaging support and the Center of Biomedical Analysis (Tsinghua University) for flow cytometry support. This work was supported by grants from the National Key R&D Program (2019YFA0508400 and 2019YFA0508403 to P.L., 2021YFF1200900 and 2018YFA0507504 to T.L.) and the Natural Science Foundation of China (32125010 and 32150023 to P.L. and 32070666 to T.L.).

Author information

Author notes

  1. These authors contributed equally: Yuan Wang, Chunyu Yu, Gaofeng Pei.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China

    Yuan Wang, Gaofeng Pei, Wen Jia & Pilong Li

  2. Tsinghua University–Peking University Joint Centre for Life Sciences, Beijing, China

    Yuan Wang, Gaofeng Pei, Wen Jia & Pilong Li

  3. Department of Biomedical Informatics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Chunyu Yu & Tingting Li

  4. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, China

    Chunyu Yu

  5. Key Laboratory for Neuroscience, Ministry of Education/National Health Commission of China, Peking University, Beijing, China

    Tingting Li

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Contributions

Y.W., C.Y., T.L. and P.L. conceived the ideas. T.L. and P.L. supervised the project. Y.W. performed all cell biology experiments and performed biochemical and genetic experiments with support from G.P. and W.J. C.Y. performed all bioinformatic analyses and image analyses. Y.W., C.Y. and G.P. performed data analyses. Y.W, C.Y. and G.P. wrote the paper. T.L. and P.L. edited the paper. All authors have commented on and approved the paper.

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Correspondence to Tingting Li or Pilong Li.

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Tsinghua University and Peking University have filed a patent application on the basis of this work. The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Cell experiments confirm phase separation of FUS-ERG.

a, SDS-PAGE analysis of MBP-EGFP-FUS-ERG purified from E. coli. b, Agarose electrophoresis analysis of purified 306-bp 25× GGAA dsDNA (labeled with cy5). c, Sequence of 306-bp dsDNA with 25× GGAA bases highlighted in red. d. Images of 40 μM EGFP-FUS-ERG mixed with 50 nM 306-bp dsDNA. The 306-bp dsDNA contained 25× GGAA and was labeled with Cy5. Experiments were performed three times with similar results. Scale bar, 10 μm. e, Representative fluorescence images of FRAP of the condensates formed by EGFP-FUS-ERG (40 μM) and 306-bp dsDNA (50 nM, 25× GGAA, labeled with Cy5). Scale bar, 2 μm. f, Quantitative analysis of the FRAP data in e. n = 6 independent measurements, data are presented as mean ± SD. g, Representative fluorescence images of FRAP for FUS-ERG and FUS-ERGmut in 293 T. A 546 nm laser with 10% power was used, and the photobleaching time was 1 second. Scale bar, 10 μm. h, Quantitative analysis of the FRAP data in g, n = 6 (for FUS-ERG) and n = 3 (for FUS-ERGmut) independent measurements, data are presented as mean ± SD. i, Representative images of FUS-ERGmut fusion proteins in 293 T treated with 10%(w/v) 1,6-hexanediol. Experiments were performed three times with similar results. Scale bar, 10 μm.

Source data

Extended Data Fig. 2 PS proteins drive phase separation in cellulo and in vitro.

a, SDS-PAGE analyses of indicated PS proteins purified from E. coli. b-f, PS proteins drive phase separation in vitro. (Top panel in b-f) Representative images and quantification of OptoIDR results of the indicated SFPQ IDR-mCherry-Cry2olig (b), MED15 IDR-mCherry-Cry2olig (c), COL17A1 IDR-mCherry-Cry2olig (d), BRD9 IDR-mCherry-Cry2olig (e), MLLT1 IDR-mCherry-Cry2olig (f). Scale bars, 20 µm. n = 29, 28, 32 and 32 independent cells per construct for SFPQ, MED15, MLLT1 and mCherry (negative control). n = 28, 20 and 36 independent cells per construct for COL17A1, BRD9 and mCherry (negative control). Data are presented as mean values +/- 95% confidence interval. (Middle panel in b-f) Confocal fluorescence images of EGFP-SFPQ IDR, EGFP-MED15 IDR, COL17A1 IDR (Alexa 488 labeled), BRD9 IDR (Alexa 488 labeled) and MLLT1 IDR (Alexa 488 labeled) at the indicated concentrations. Experiments were performed three times with similar results. Scale bars, 10 μm. (bottom panel in b-f) FRAP and quantitative analysis of the condensates formed by EGFP-SFPQ IDR (10 μM), EGFP-MED15 IDR (10 μM), COL17A1 IDR (100 μM), BRD9 IDR (150 μM), MLLT1 IDR (25 μM). The FRAP data are from different independent measurements: for EGFP-SFPQ IDR (n = 8), EGFP-MED15 IDR (n = 9), COL17A1 IDR (n = 6), BRD9 IDR (n = 9) and MLLT1 IDR (n = 12). Data are presented as mean ± SD. Scale bars, 2 μm.

Source data

Extended Data Fig. 3 Validations of selected PS-DBD fusion proteins.

Schematic diagram of indicated PS, DBD and PS-DBD fusion proteins and disorder prediction (left in each dotted box), the red rectangle indicates DBD. Live-cell images of HeLa cells that ectopically express indicated mCherry-PS-DBD and its mutated DNA-binding domain form mCherry-PS-DBDmut (right in each dotted box). DNA is stained by Hoechst 33342. Experiments were performed three times with similar results. Scale bars, 10 μm.

Extended Data Fig. 4 Cell experiments prove phase separation of PS-DBD fusion proteins.

a-f, Representative data of FRAP and 1,6-HD treatment assay for PS-DBD fusion proteins. SFPQ-TFE3 in a, SFPQ-TFE3mut in b, MED15-TFE in c, MED15-TFE3mut in d, EWS-FLI1 in e, EWS-FLI1mut in f. (Left panel) Representative images of FRAP for PS-DBD/PS-DBDmut fusion proteins in 293T. A 546 nm laser with 10% power was used, and the photobleaching time was 1 second. Scale bar, 10 μm. (Middle panel) Corresponding quantitative data of FRAP. (Right panel) Representative images of PS-DBD/PS-DBDmut fusion proteins in 293T treated with 10%[w/v] 1,6-hexanediol. Scale bar, 10 μm. For the FRAP in a-f, data are from different independent measurements: n = 4 for a, n = 3 for b, n = 6 for c, n = 6 for d, n = 8 for e, n = 3 for f. Data are presented as mean ± SD. For the 1,6-hexanediol results in a-f, all experiments were performed three times with similar results.

Source data

Extended Data Fig. 5 PS-DBD fusions cause aberrant target gene expression.

a, GSEA analysis of DBD targets for PS-DBD fusion tumor samples in the TCGA database. For each sample with a PS-DBD fusion, samples of the same tumor type without this fusion were selected as the background set. b, GSEA analysis of FLI1 targets for EWS-FLI1 fusion cell lines in the CCLE database. Six cell lines with EWS-FLI1 fusions which significantly increase the downstream transcription are shown; cell lines from bone without EWS-FLI1 fusion were used as background.

Extended Data Fig. 6 Representative hits compounds screened by DropScan.

a, DropScan workflow. DropScan uses U2OS/mCherry-FUS-ERGmut cells to screen small molecules from the APExBio Anti-cancer compound library Plus. The system monitors the fraction of cells with droplets in a time-series manner. (b-i), Characteristics of 8 representative hit compounds.Corresponding images and quantifications before and after 6 hours treatment are shown for CHIR-124 (b), GDC-0941 (c), EMD-1214063 (d), AZD-9291 (e), CX-6258 (f), UNC 0631 (g), Pelitinib (h) and Ursolic acid (i). Scale bar, 50 μm.

Source data

Extended Data Fig. 7 LY2835219 decreases the amounts of condensate and activates lysosome.

a, Live-cell images of U2OS/mCherry-FUS-ERGmut cells treated by LY2835219 with indicated concentration. Scale bar, 50 μm. b, Lysotracker Green staining images of U2OS cells after 4 hours treatment with 7.5 μM LY2835219. Experiments were performed three times with similar results. Scale bars, 50 μm. c, Western Blotting analysis for U2OS/mCherry-FUS-ERGmut treated with 7.5 μM LY2835219 for indicated time. Fusion proteins were detected with anti-mCherry antibodies, and GAPDH was used as a loading control. Experiments were performed three times with similar results. GAPDH was used as a loading control. Experiments were performed three times with similar results.

Source data

Extended Data Fig. 8 PSmut of FUS-ERG failed to phase separation and destroy the transcription activation.

a, Live-cell images of EWS-FLI1, FUS-ERG and their PSmut and DBDmut fusion proteins in 293 T cells. Experiments were performed three times with similar results. Scale bars, 10 μm. b, Results of the dual luciferase reporter assay in cells treated with or without 7.5 μM LY2835219 for 6hs. HEK293T cells were transfected with the dual luciferase reporter plasmid and the expression vector for the mCherry-FUS-ERG and mCherry-FUS-ERG mutant fusion protein, mCherry without fusion protein as negative control. Six repeats per group. Two-tailed independent samples t-test. Data are presented as mean ± SD. c, Schematic depicting how PS-DBD fusion proteins drive aberrant phase separation in the related cancers. Under normal conditions, genes undergo typical expression; when a chromosomal rearrangement occurs to create an in-frame PS-BDB fusion, phase separation at the target DNA sites leads to aberrant gene expression, which is correlated with cancer. Small molecules that dissolve the phase-separated condensates can rescue the abnormal expression.

Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Landscape of PS–DBD fusions in public databases.

Supplementary Table 2

Screening results of the DropScan pipeline.

Supplementary Table 3

Transcriptome profiles of A673 cell lines before and after treatment with LY2835219.

Supplementary Table 4

Transcriptome profiles of U2OS/mCherry–EWS–FLI1 cell lines before and after treatment with LY2835219, U2OS/mCherry, U2OS/mCherry–EWS–FLI1mut and U2OS/mCherry–EWS(YS37)–FLI1 cell lines.

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Wang, Y., Yu, C., Pei, G. et al. Dissolution of oncofusion transcription factor condensates for cancer therapy. Nat Chem Biol 19, 1223–1234 (2023). https://doi.org/10.1038/s41589-023-01376-5

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