The clear cell sarcoma functional genomic landscape
Emanuele Panza, … , Mario R. Capecchi, Kevin B. Jones
Emanuele Panza, … , Mario R. Capecchi, Kevin B. Jones
Published June 22, 2021
Citation Information: J Clin Invest. 2021;131(15):e146301. https://doi.org/10.1172/JCI146301.
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Research Article Genetics Oncology

The clear cell sarcoma functional genomic landscape

Abstract

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP–induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.

Authors

Emanuele Panza, Benjamin B. Ozenberger, Krystal M. Straessler, Jared J. Barrott, Li Li, Yanliang Wang, Mingchao Xie, Anne Boulet, Simon W.A. Titen, Clinton C. Mason, Alexander J. Lazar, Li Ding, Mario R. Capecchi, Kevin B. Jones

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Figure 3

Exome sequencing of EWSR1-ATF1 expression–initiated mouse tumors reveals that no secondary alterations are strictly required to complete sarcomagenesis.

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Exome sequencing of EWSR1-ATF1 expression–initiated mouse tumors reveals...
(A) Schematic illustrating CreERT2 engineered at the Bmi1 locus in conjunction with Cre-inducible human EWSR1-ATF1 engineered at the Rosa26 locus. Cre recombinase removes the neoR-STOP cassette to induce expression of EWSR1-ATF1, and the GFP reporter confirms EWSR1-ATF1 expression. (B) Tamoxifen injection induces CreERT2 activity in Bmi1-expressing stem cells (blue), and then in turn, Cre recombinase induces Rosa26-mediated expression of EWSR1-ATF1 (EA1). (C) Time course showing age at injection and age at tumor harvesting (n = 6). (D) Summary of total number of tumors detected and harvested per mouse (n = 6). (E) Exome sequencing of 34 mouse tumors, with 1 tumor’s exome presented on each line, clustered by host mouse, and each variant allele denoted by a circle whose size corresponds to the VAF. All variants with a VAF of greater than 0.25 are also identified by corresponding colors in the list of gene symbols and protein amino acid substitutions below. (F) RNA-Seq rendered expression levels relative to Gapdh for the 8 genes with variants present at fractions higher than 0.4. The level of Mitf expression is included for reference. (G) Exome-wide CNV was inferred from exome sequencing data, as shown for 12 mouse tumors. Normal diploidy is represented by the x axis crossing at a value of y = 2.