Functional Genomics of Human Clear Cell Sarcoma

Functional Genomics of Human Clear Cell Sarcoma
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Background

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma of children and young adults (mean age 22 years). Up to 30% of patients present metastatic disease at diagnosis and overall the 10-year survival rate for patients with CCS have been reported as only 33%. Relapse with metastases results in a significant unmet clinical need as surgery is often not possible and effective targeted therapies are not yet available (1).

CCS are characterized by reciprocal translocation t(12;22) or t(2;22) resulting in EWSR1-ATF1 or EWSR1-CREB1 fusion genes, respectively (1). They produce chimeric transcription factor that is known to dysregulate MITF (melanocyte-inducing transcription factor), that was suggested as a key driver of CCS progression (2). Nevertheless, additional mutations most likely contribute to CCS development and/or can be used as therapeutic targets for the treatment (3,4).

In this study, we undertook a multi-pronged approach to understand the biology of the disease, to identify key molecular alternation and to use this knowledge to establish effective treatment of CCS. In short we conducted 1/ genomic and transcriptomic landscape analysis, using DNA whole exome- and RNA sequencing; 2/ validation of putative targets; 3/development and evaluation of potential therapeutics for CCS (Fig.1).

Figure 1. Overview of studies performed.

Results

DNA whole exome sequencing identified recurrent (homozygous) loss of genes involved in cell cycle checkpoint (e.g. ATM, CHEK1, CHEK2), DNA double-strand break repair (MRE11, RAD50) or DNA mismatch repair genes (PMS2, MLH1, MSH2, MSH6) with corresponding low gene expression by RNA sequencing. At the same time genomic instability, defined by tumor mutational burden of non-silent mutations, was low to intermediate. Multicopy gains affected PABPC1, GNAS and RPLP0, with high expression level of >500 transcripts per million (TPM) at the transcriptome level. Furthermore, MYC and MITF presented with gene amplification, with confirmed high level of gene expression. Genes overexpressed but not necessarily amplified included RPL7A, FN1 and ALDOA.

Unsupervised clustering identified HER3 ad EGFR signaling pathway genes as enriched in gastrointestinal (GI) CCS. Notably, HER3 was also expressed in non-GI CCS tumors, although with a larger range of expression values (Fig. 2A). HER3 presence was confirmed at the protein level in CCS cell lines using immunoblotting (Fig. 2b). Regardless, HER3 expressing cell lines were generally insensitive to AZD8931 (HER3 small molecule inhibitor) especially compared to EKB-569 (EGFR/HER2 inhibitor) in vitro, suggesting CCS cells may not rely on HER3 for autonomous cell survival. In contrast, patritumab deruxtecan (HER3-DXd) an antibody-drug conjugate (ADC) consisting of a HER3 antibody attached to a topoisomerase I inhibitor payload, showed a dose-dependent efficacy in vitro.

Figure 2. Expression and function of HER3. (A) RNA expression of EGFR, HER2 and HER3 genes in gastrointestinal (GI) and non-GI CCS tumors presented as number of transcripts per million (TPM). (B) Western blot of HER3 for available CCS cell lines, with MCF-7 breast cancer cell line as positive control.

In the next step the molecular evaluation of CCS was complemented with a targeted drug screening of available CCS cell lines, using 61 pre-clinical and clinical compounds, selected based on the relevance to biological pathways implicated in sarcoma and targets currently under investigation as therapeutic mechanisms in sarcoma. None of the tested agents were selective for CCS vs Ewing sarcoma or normal cell lines in 72 hour cell viability assay, with a limited activity against CCS cells showed by CHK1 inhibitors (LY2606368, CHIR-124).

Finally, we initiated a drug development program to inhibit CREB1 and ATF1 mediated transcript, knowing that EWSR1-ATF1 fusion is considered a CCS driver. Based on the CREB1-TORC2 crystal structure a total of 90 compounds were identified as potential antagonists (Fig. 3a). In a luciferase-based CRE transcription reporter assay, 8 hit compounds were validated for dose-response activity (Fig. 3 b-c).

Figure 3. Atomwise screen and validations for CREB1 antagonists. (A) Ribbon structure of CREB1 docking site and residues. A white sphere for compound docking is shown at the CREB1-CRTC2 interface. (B) Chemical structure of 6G and 7F. (C) The unchanged morphology in KAS cells exposed to the respective compounds.

In conclusion: our study of the genomic, transcriptomic and chemical biology landscape represent a multi-dimensional CCS characterization, available for the future investigation, target identification and drug development that would ultimately lead to the improvement of the treatment of patients with CCS.

References

  1. 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 May;63(5):416–23.
  2. 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 Jun;9(6):473–84.
  3. Panza E, Ozenberger BB, Straessler KM, Barrott JJ, Li L, Wang Y, et al. The clear cell sarcoma functional genomic landscape. J Clin Invest. 2021;131(15).
  4. 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 (Basel). 2021 Dec 1;13(23).

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Go to the profile of Charles Keller
11 months ago

Aga, we are tremendously grateful to have collaborated with you on this study.  Charles * the cc-TDI team. 

Go to the profile of Agnieszka Wozniak
11 months ago

Thanks Charles! It was a pleasure!

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