Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Genetics and Genomics

Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma



Systemic therapy for metastatic clear cell sarcoma (CCS) bearing EWSR1-CREB1/ATF1 fusions remains an unmet clinical need in children, adolescents, and young adults.


To identify key signaling pathway vulnerabilities in CCS, a multi-pronged approach was taken: (i) genomic and transcriptomic landscape analysis, (ii) integrated chemical biology interrogations, (iii) development of CREB1/ATF1 inhibitors, and (iv) antibody-drug conjugate testing (ADC). The first approach encompassed DNA exome and RNA deep sequencing of the largest human CCS cohort yet reported consisting of 47 patient tumor samples and 8 cell lines.


Sequencing revealed recurrent mutations in cell cycle checkpoint, DNA double-strand break repair or DNA mismatch repair genes, with a correspondingly low to intermediate tumor mutational burden. DNA multi-copy gains with corresponding high RNA expression were observed in CCS tumor subsets. CCS cell lines responded to the HER3 ADC patritumab deruxtecan in a dose-dependent manner in vitro, with impaired long term cell viability.


These studies of the genomic, transcriptomic and chemical biology landscape represent a resource ‘atlas’ for the field of CCS investigation and drug development. CHK inhibitors are identified as having potential relevance, CREB1 inhibitors non-dependence of CCS on CREB1 activity was established, and the potential utility of HER3 ADC being used in CCS is found.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Graphical Abstract.
Fig. 2: CCS Histology.
Fig. 3: Genetic Summary of Next Generation DNA and RNA sequencing.
Fig. 4: Potential Targets Defined by WGCNA Eigengene Modules.
Fig. 5: Expression and Functional Inhibition of HER3.
Fig. 6: Atomwise Screen and Validations for CREB1 Antagonists.

Data availability

DNA and RNA sequencing data performed for this manuscript is available for access through the EGA repository as Dataset: EGAD00001008611, Study: EGAS00001006072.


  1. Green C, Spagnolo DV, Robbins PD, Fermoyle S, Wong DD. Clear cell sarcoma of the gastrointestinal tract and malignant gastrointestinal neuroectodermal tumour: distinct or related entities? A review. Pathology. 2018;50:490–8.

    Article  PubMed  Google Scholar 

  2. Dim DC, Cooley LD, Miranda RN. Clear cell sarcoma of tendons and aponeuroses: a review. Arch Pathol Lab Med. 2007;131:152–6.

    Article  CAS  PubMed  Google Scholar 

  3. Wang H, Wang L, Zhang G, Lu C, Chu H, Yang R, et al. MALAT1/miR-101-3p/MCL1 axis mediates cisplatin resistance in lung cancer. Oncotarget 2018;9:7501–12.

    Article  PubMed  Google Scholar 

  4. Kosemehmetoglu K, Folpe AL. Clear cell sarcoma of tendons and aponeuroses, and osteoclast-rich tumour of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: a review and update. J Clin Pathol. 2010;63:416–23.

    Article  PubMed  Google Scholar 

  5. Wang WL, Mayordomo E, Zhang W, Hernandez VS, Tuvin D, Garcia L, et al. Detection and characterization of EWSR1/ATF1 and EWSR1/CREB1 chimeric transcripts in clear cell sarcoma (melanoma of soft parts). Mod Pathol. 2009;22:1201–9.

    Article  CAS  PubMed  Google Scholar 

  6. Yokoyama S, Woods SL, Boyle GM, Aoude LG, MacGregor S, Zismann V, et al. A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma. Nature 2011;480:99–103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Davis IJ, Kim JJ, Ozsolak F, Widlund HR, Rozenblatt-Rosen O, Granter SR, et al. Oncogenic MITF dysregulation in clear cell sarcoma: defining the MiT family of human cancers. Cancer Cell. 2006;9:473–84.

    Article  CAS  PubMed  Google Scholar 

  8. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005;436:117–22.

    Article  CAS  PubMed  Google Scholar 

  9. Panza E, Ozenberger BB, Straessler KM, Barrott JJ, Li L, Wang Y, et al. The clear cell sarcoma functional genomic landscape. J Clin Investig. 2021;131:e146301.

  10. Bayat Mokhtari R, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, et al. Combination therapy in combating cancer. Oncotarget. 2017;8:38022–43.

    Article  PubMed  Google Scholar 

  11. Wang J, Thway K. Clear cell sarcoma-like tumor of the gastrointestinal tract: an evolving entity. Arch Pathol Lab Med. 2015;139:407–12.

    Article  PubMed  Google Scholar 

  12. Segawa K, Sugita S, Aoyama T, Kubo T, Asanuma H, Sugawara T, et al. Detection of specific gene rearrangements by fluorescence in situ hybridization in 16 cases of clear cell sarcoma of soft tissue and 6 cases of clear cell sarcoma-like gastrointestinal tumor. Diagn Pathol. 2018;13:73.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Lee CJ, Modave E, Boeckx B, Stacchiotti S, Rutkowski P, Blay JY, et al. Histopathological and Molecular Profiling of Clear Cell Sarcoma and Correlation with Response to Crizotinib: An Exploratory Study Related to EORTC 90101 “CREATE” Trial. Cancers. 2021;13:6057.

  14. Möller E, Praz V, Rajendran S, Dong R, Cauderay A, Xing YH, et al. EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma. Nat Commun. 2022;13:2267.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Barlaam B, Anderton J, Ballard P, Bradbury RH, Hennequin LFA, Hickinson DM, et al. Discovery of AZD8931, an Equipotent, Reversible Inhibitor of Signaling by EGFR, HER2, and HER3 Receptors. ACS Med Chem Lett. 2013;4:742–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tse AN, Rendahl KG, Sheikh T, Cheema H, Aardalen K, Embry M, et al. CHIR-124, a novel potent inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I poisons in vitro and in vivo. Clin Cancer Res. 2007;131:591–602.

    Article  Google Scholar 

  17. Xie F, Li BX, Xiao X. Design, synthesis and biological evaluation of regioisomers of 666-15 as inhibitors of CREB-mediated gene transcription. Bioorg Med Chem Lett. 2017;27:994–8.

    Article  CAS  PubMed  Google Scholar 

  18. Egawa Y, Saigo C, Kito Y, Moriki T, Takeuchi T. Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma. PLoS ONE. 2018;13:e0198940.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Woods AD, Purohit R, Mitchell LC, Collier JR, Collier KA, Lathara M, et al. Metastatic pediatric sclerosing epithelioid fibrosarcoma. Cold Spring Harb Mol Case Stud. 2021;7:a006093.

  20. Li J, Chen C, Liu W, Liu S, Hu W, Gao X, et al. Whole-exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets. Clin Transl Med. 2021;11:e640.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Jones RL, Constantinidou A, Thway K, Ashley S, Scurr M, Al-Muderis O, et al. Chemotherapy in clear cell sarcoma. Med Oncol. 2011;28:859–63.

    Article  PubMed  Google Scholar 

  22. Schöffski P, Wozniak A, Stacchiotti S, Rutkowski P, Blay JY, Lindner LH, et al. Activity and safety of crizotinib in patients with advanced clear-cell sarcoma with MET alterations: European Organization for Research and Treatment of Cancer phase II trial 90101 “CREATE”. Ann Oncol. 2019;30:344.

    Article  PubMed  Google Scholar 

  23. Wagner AJ, Goldberg JM, Dubois SG, Choy E, Rosen L, Pappo A, et al. Tivantinib (ARQ 197), a selective inhibitor of MET, in patients with microphthalmia transcription factor-associated tumors: results of a multicenter phase 2 trial. Cancer. 2012;118:5894–902.

    Article  CAS  PubMed  Google Scholar 

  24. Schaefer KL, Brachwitz K, Wai DH, Braun Y, Diallo R, Korsching E, et al. Expression profiling of t(12;22) positive clear cell sarcoma of soft tissue cell lines reveals characteristic up-regulation of potential new marker genes including ERBB3. Cancer Res. 2004;64:3395–405.

    Article  CAS  PubMed  Google Scholar 

  25. Beleaua MA, Jung I, Braicu C, Milutin D, Gurzu S SOX11, SOX10 and MITF Gene Interaction: A Possible Diagnostic Tool in Malignant Melanoma. Life. 2021;11:281.

  26. Faloon PW, Bennion M, Weiner WS, Smith RA, Wurst J, Weiwer M, et al. A Small Molecule Inhibitor of the MITF Molecular Pathway. Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD); 2010.

  27. Bresnick AR. S100 proteins as therapeutic targets. Biophys Rev. 2018;10:1617–29.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Jänne PA, Baik C, Su WC, Johnson ML, Hayashi H, Nishio M, et al. Efficacy and Safety of Patritumab Deruxtecan (HER3-DXd) in EGFR Inhibitor-Resistant, EGFR-Mutated Non-Small Cell Lung. Cancer Cancer Discov. 2022;12:74–89.

    Article  PubMed  Google Scholar 

  29. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinform. 2008;9:559.

    Article  Google Scholar 

  30. Zhang B, Horvath S. A general framework for weighted gene co-expression network analysis. Stat Appl Genet Mol Biol. 2005;4:Article17.

    Article  PubMed  Google Scholar 

  31. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1–13.

    Article  PubMed  Google Scholar 

  32. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R Stat Soc Ser B. 1995;57:289–300.

    Google Scholar 

  33. Song Y, Zhai L, Valencia Swain J, Chen Y, Wang P, Chen L, et al. Structural Insights into the CRTC2-CREB Complex Assembly on CRE. J Mol Biol. 2018;430:1926–39.

    Article  CAS  PubMed  Google Scholar 

  34. Schroedl S. Current methods and challenges for deep learning in drug discovery. Drug Disco Today Technol. 2019;32–33:9–17.

    Article  Google Scholar 

  35. Hiraga H, Nojima T, Abe S, Yamashiro K, Yamawaki S, Kaneda K, et al. Establishment of a new continuous clear cell sarcoma cell line. Morphological and cytogenetic characterization and detection of chimaeric EWS/ATF-1 transcripts. Virchows Arch. 1997;431:45–51.

    Article  CAS  PubMed  Google Scholar 

  36. Liao SK, Perng YP, Lee LA, Chang KS, Lai GM, Wong E, et al. Newly established MST-1 tumour cell line and tumour-infiltrating lymphocyte culture from a patient with soft tissue melanoma (clear cell sarcoma) and their potential applications to patient immunotherapy. Eur J Cancer. 1996;32A:346–56.

    Article  CAS  PubMed  Google Scholar 

  37. Epstein AL, Martin AO, Kempson R. Use of a newly established human cell line (SU-CCS-1) to demonstrate the relationship of clear cell sarcoma to malignant melanoma. Cancer Res. 1984;44:1265–74.

    CAS  PubMed  Google Scholar 

  38. Moritake H, Sugimoto T, Asada Y, Yoshida MA, Maehara Y, Epstein AL, et al. Newly established clear cell sarcoma (malignant melanoma of soft parts) cell line expressing melanoma-associated Melan-A antigen and overexpressing C-MYC oncogene. Cancer Genet Cytogenet. 2002;135:48–56.

    Article  CAS  PubMed  Google Scholar 

  39. Hakozaki M, Tamura H, Dobashi Y, Yoshida A, Kato K, Tajino T, et al. Establishment and Characterization of a Novel Human Clear-cell Sarcoma of Soft-tissue Cell Line, RSAR001, Derived from Pleural Effusion of a Patient with Pleural Dissemination. Anticancer Res. 2018;38:5035–42.

    Article  CAS  PubMed  Google Scholar 

  40. Jishage M, Fujino T, Yamazaki Y, Kuroda H, Nakamura T. Identification of target genes for EWS/ATF-1 chimeric transcription factor. Oncogene 2003;22:41–9.

    Article  CAS  PubMed  Google Scholar 

  41. Nakai T, Imura Y, Tamiya H, Yamada S, Nakai S, Yasuda N, et al. Trabectedin is a promising antitumor agent potentially inducing melanocytic differentiation for clear cell sarcoma. Cancer Med. 2017;6:2121–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Schaefer KL, Wai DH, Poremba C, Korsching E, van Valen F, Ozaki T, et al. Characterization of the malignant melanoma of soft-parts cell line GG-62 by expression analysis using DNA microarrays. Virchows Arch. 2002;440:476–84.

    Article  CAS  PubMed  Google Scholar 

  43. Outani H, Tanaka T, Wakamatsu T, Imura Y, Hamada K, Araki N, et al. Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS. BMC Cancer. 2014;14:455.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Takenouchi T, Ito K, Kazama T, Ito M. Establishment and characterization of a clear-cell sarcoma (malignant melanoma of soft parts) cell line. Arch Dermatol Res. 1994;286:254–60.

    Article  CAS  PubMed  Google Scholar 

  45. Sonobe H, Furihata M, Iwata J, Ohtsuki Y, Mizobuchi H, Yamamoto H, et al. Establishment and characterization of a new human clear-cell sarcoma cell-line, HS-MM. J Pathol. 1993;169:317–22.

    Article  CAS  PubMed  Google Scholar 

  46. Sakumoto M, Oyama R, Takahashi M, Takai Y, Kito F, Shiozawa K, et al. Establishment and proteomic characterization of patient-derived clear cell sarcoma xenografts and cell lines. Vitr Cell Dev Biol Anim. 2018;54:163–76.

    Article  CAS  Google Scholar 

  47. Crnalic S, Panagopoulos I, Boquist L, Mandahl N, Stenling R, Löfvenberg R. Establishment and characterisation of a human clear cell sarcoma model in nude mice. Int J Cancer. 2002;101:505–11.

    Article  CAS  PubMed  Google Scholar 

  48. Brown AD, Lopez-Terrada D, Denny C, Lee KA. Promoters containing ATF-binding sites are de-regulated in cells that express the EWS/ATF1 oncogene. Oncogene 1995;10:1749–56.

    CAS  PubMed  Google Scholar 

Download references


We thank Drs. William Tap, Robert Maki, Lia Gore, Carrye Cost, Margaret Macy, and Robin Jones for assistance with therapeutic agent selection for the chemical screen. We are grateful for technical assistance of Kenneth Crawford. We are grateful to Daiichi Sankyo who provided materials for ADC studies. We thank Drs. Miguel Rivera, Emely Möller and Nicolò Riggi for histone track data related to their publication.


This work was supported by The Sara’s Cure Foundation, Rocker Collier Foundation and Golf Fights Cancer. We thank the Sam Day Foundation for funding for patient sample processing & sequencing through the CuReFast program at DEF gratefully acknowledges support from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and NIH Grants 5R01AR043369–25, 5R01CA222871–04, 5P01CA163222–08, and 5R01AR072304–05.

Author information

Authors and Affiliations



NEB, AW, LRB, SVR, PS and CK designed the studies. AW, JL, TK, XX, DEF, EI, IM, ALB, ERR, HM, TN, KT, RLJ, PHH, JW, NG, ILA and PS provided reagents. SVR, LRB, SC, BMS and JMG performed the experiments. SVR, JMG, ML, HW, ADW, KT, NEB, PS, MKS, KN, GS and CK analyzed the data. CK supervised all studies. All authors critically reviewed the report and approved the final version.

Corresponding authors

Correspondence to Paul H. Huang, Noah E. Berlow or Charles Keller.

Ethics declarations

Competing interests

CK has sponsored research agreements with Eli Lily, Roche-Genentech and Cardiff Oncology as well as recent collaborations with Novartis, and is co-founder of Tio Companies. Artisan Biopharma is a wholly-owned subsidiary of cc-TDI. PS has received honoraria from Blueprint Medicines, consults with Deciphera, Ellipses Pharma, Blueprint Medicines,Transgene, Exelixis, Boehringer Ingelheim, Ysios Capital, Studiecentrum voor Kernenergie, Modus Outcomes, Curio Science, SQZ Biotechnology, CRT Pioneer Fund LP, Adcendo, PharmaMar, Merck Healthcare KGaA, Advance Medical/Teladoc Health, and receives research funding from CoBioRes NV, Eisai, G1 Therapeutics, PharmaMar, Genmab, Merck, Sartar Therapeutics, and ONA Therapeutics. DEF has a financial interest in Soltego, a company developing salt inducible kinase inhibitors for topical skin-darkening treatments that might be used for a broad set of human applications. The interests of DEF were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. NEB is founder of First Ascent Biomedical.

Ethics approval

Under institutional review board/human studies ethics committee approvals, de-identified CCS tissue samples were collected from the CuReFast Biobank (Advarra, protocol # cc-TDI-IRB-1), The Royal Marsden Hospital, Universitaire Ziekenhuizen Leuven (University Hospitals Leuven) and the European Organization for Research and Treatment of Cancer (EORTC) 90101 CREATE study [13]. All patients gave written informed consent under approved institutional ethics board protocols (REB# 01–0138-U) and studies were conducted according to the Declaration of Helsinki.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rasmussen, S.V., Wozniak, A., Lathara, M. et al. Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma. Br J Cancer 128, 1941–1954 (2023).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links