Generation of human embryonic stem cell models to exploit the EWSR1-CREB fusion promiscuity as a common pathway of transformation in human tumors

Our plain-English summary of Generation of human embryonic stem cell models to exploit the EWSR1-CREB fusion promiscuity as a common pathway of transformation in human tumors, by F. Vanoli, B. Meskauskaite, L. Herviou, et al (Oncogene, 2021).

The study

• Authors: F. Vanoli, B. Meskauskaite, L. Herviou, W. Mallen, Y.S. Sung, Y. Fujisawa, L. Zhang, S. Simon, D. Huangfu, M. Jasin, C.R. Antonescu
• Journal: Oncogene
• Published: 2021;40(32):5095-5104
• Cited by: 13 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1 fusions with CREB1 and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1 and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1 fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1 fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.

© 2021. The Author(s), under exclusive licence to Springer Nature Limited.

In plain English

This was a laboratory study, not a study of people exposed to asbestos. The authors noted that EWSR1 fusions with CREB1 and ATF1 occur in several tumour types, including sarcomas, carcinomas and young adult mesothelioma. Using CRISPR-Cas9 and homology-directed repair, they engineered human embryonic stem (hES) cells carrying EWSR1-CREB1, EWSR1-ATF1 and EWSR1-WT1 translocations that could be switched on with Cre recombinase, and they re-analysed gene expression in human angiomatoid fibrous histiocytoma (AFH), clear cell sarcoma (CCS) and gastrointestinal clear cell sarcoma (GI-CCS) tumour samples. Turning on EWSR1-CREB1 or the in-frame EWSR1-ATF1 fusion in hES cells reproduced the AFH and GI-CCS core gene signatures but impaired proliferation and/or survival; the fusion transcripts and signatures were undetectable by about day 11 or 12, with cells not expressing the fusion overgrowing the culture, and deleting TP53 did not rescue them. In hES-derived mesenchymal progenitors both fusions were tolerated for about a month, and for EWSR1-CREB1 deleting TP53 extended fusion expression and the AFH signature to about seven weeks. The authors concluded such isogenic lines can help dissect how single fusions drive tumours.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experimental study: CRISPR-Cas9 genome editing to create conditional oncogenic fusion genes in human embryonic stem cells and derived mesenchymal progenitors, combined with gene expression profiling of human tumour samples
• Who was studied: Human embryonic stem cells (WA01, H1), hES-derived mesenchymal progenitor cells and HEK293 cells; plus human tumour samples (3 AFH and 4 CCS cases on Affymetrix array, compared with 44 other sarcomas and 6 normal tissues, and additional AFH, CCS and GI-CCS cases by RT-PCR and RNA sequencing), at Memorial Sloan Kettering Cancer Center
• Exposure: Not an asbestos or other environmental exposure: the manipulated variable was conditional expression of engineered chromosomal translocations EWSR1(ex7)-CREB1(ex7), EWSR1(ex7)-ATF1(ex4), EWSR1(ex7)-ATF1(ex5) and EWSR1(ex7)-WT1(ex8), with or without TP53 deletion
• Main results:
  • In hES cells, inducing the EWSR1(ex7)-CREB1(ex7) fusion upregulated SGK1 and MXRA5, while PMEL, SOX10, SLC7A5 and DUSP4 were not significantly increased
  • In hES cells, the in-frame EWSR1(ex7)-ATF1(ex5) fusion upregulated SGK1, MXRA5, SOX10 and DUSP4 but not PMEL or SLC7A5; the out-of-frame ex4 fusion produced no transcriptional changes
  • Translocation-positive clone recovery in hES cells for EWSR1(ex7)-CREB1(ex7): with DNA-PK inhibitor, 13 of 146 hygromycin-positive clones were positive by out-in and in-out PCR, 2 also by out-out PCR; with DMSO, 6 of 144 clones, none out-out positive
  • In hES cells the EWSR1-CREB1 fusion and AFH signature genes were present at days 4 and 7 after Cre but absent at day 11; the authors attributed this to impaired proliferation and/or survival with overgrowth by non-expressing cells, and none of 5 single-cell clones had undergone Cre-mediated recombination
  • Deleting TP53 in hES cells did not restore viability, and Annexin V positive cells increased even in TP53-null cells
  • hES-derived mesenchymal progenitor cells expressing EWSR1-CREB1 remained viable about one month and induced SGK1 and MXRA5; a similar observation was made for cells expressing EWSR1-ATF1 and the GI-CCS signature
  • In hES-MP cells with the EWSR1-CREB1 translocation, TP53 deletion maintained fusion transcripts and the AFH core gene signature for about 7 weeks, a partial rescue of lethality
  • HEK293 cells tolerated both fusions but did not reproduce the AFH, CCS or GI-CCS expression signatures
  • In hES-MP cells expressing EWSR1-WT1, no AFH, CCS or GI-CCS signature genes were upregulated, while PDGFA was upregulated
• The authors' conclusion: The authors concluded that combining CRISPR-Cas9 with homology-directed repair in hES cells, differentiation to an appropriate cell type and conditional fusion expression provides a flexible system, with potential to investigate the cell of origin in human neoplasia lacking a clear line of differentiation, including translocation-associated sarcomas
• Limitations the authors state:
  • Fusion expression alone did not cause cellular transformation in the soft agar assay, and colonies formed in the TP53-null background whether or not the fusion was expressed, so a specific role for the fusion could not be determined
  • Existing transcriptional profiling data for GI-CCS were limited, with only one previously analysed EWSR1-CREB1 case
  • Fusion expression in hES-MP cells still declined after several weeks, and further TP53-dependent or independent functions remain to be uncovered to fully rescue proliferation or induce transformation

Topics

Medical terms in this study, explained in our Lexicon: STEM, Translocation, Sarcoma, Carcinoma, Mesothelioma, Tumorigenesis, Discovery.

Related studies

• A Subset of Malignant Mesotheliomas in Young Adults Are Associated With Recurrent EWSR1/FUS-ATF1 Fusions — P. Desmeules, P. Joubert, et al. (2017)
• Continuous exposure to chrysotile asbestos can cause transformation of human mesothelial cells via HMGB1 and TNF-α signaling — F. Qi, G. Okimoto, et al. (2013)
• Asbestos promotes morphological transformation and elevates expression of a gene family invariably induced by tumor promoters in C3H/10T1/2 cells — C.L. Parfett, R. Pilon, et al. (1996)
• Hippo pathway gene mutations in malignant mesothelioma: revealed by RNA and targeted exon sequencing — A. Miyanaga, M. Masuda, et al. (2015)
• RNA sequencing identifies fusion of the EWSR1 and YY1 genes in mesothelioma with t(14;22)(q32;q12) — I. Panagopoulos, J. Thorsen, et al. (2013)
• Neoplastic-like transformation effect of single-walled and multi-walled carbon nanotubes compared to asbestos on human lung small airway epithelial cells — L. Wang, T.A. Stueckle, et al. (2014)

Browse more research in Oncogene, or return to medical research.