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 4

Cre-mediated chromosomal translocation induces sarcomagenesis in the mouse.

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Cre-mediated chromosomal translocation induces sarcomagenesis in the mou...
(A) Schematic representation of the loxP sites targeted to Ewsr1 intron 7 and Atf1 intron 4, as well as the 2 products of Cre-mediated chromosomal translocation. (B) Schematics and Kaplan-Meier survival plots of tumorigenesis in mice heterozygous for Ewsr1-loxP and Atf1-loxP, induced by 3 different Cre recombinase delivery methods: a knockin allele (HprtCre), a transgenic allele (Prx1Cre), and injection of Cre recombinase protein (TATCre). (C) Gross photo of a hind-limb Prx1Cre-induced tumor (arrowhead) forming in a mouse. Scale bar: 10 mm. (D) Magnetic resonance images of HprtCre- or TATCre-induced tumors (indicated by arrowheads) forming in the thigh, dorsal pelvis, thigh, and both the pelvis and contralateral thigh of mice (upper panels, T2-weighted; lower panels, proton density–weighted). (E) Representative H&E-stained photomicrographs of histological sections of tumors from translocation (top panels), Rosa26-EA1–expressing (middle panels), and human CCS tumors (below), with (from left to right) short spindle cell, myxoid, and clear cell morphologies. Scale bars: 10 μm. (F) Schematic of the PCR amplification strategy with a schematic of primers that amplify across each translocation site, tested in genomic tumor DNA from a TATCre-induced tumor, with WT and lox alleles as well as each translocation product detected by specified primer combinations. ER, Ewsr1 reverse; EF, Ewsr1 forward; AF, Atf1 forward; AR, Atf1 reverse. (G) Graph showing the RNA-Seq–determined expression (in fragments per kilobase per million reads [FPKM]) of 2 melanocytic marker genes, with 8 HprtCre-induced translocation tumors and their mean indicated in black and the mean of 13 EA1-induced tumors in gray.