Clear Cell Sarcoma Cell Research Articles

Abstract 647: FDA approved library screen revealed Ewing and clear cell sarcomas have increased sensitivity to filanesib over other cancer types

Abstract There is a compelling need for the development of more effective and less toxic therapies for patients with Ewing Sarcoma (ES) and Clear Cell Sarcoma (CCS). ES and CCS are characterized by chromosomal translocations which result in two distinct fusion proteins: EWS-ATF1 (t(12;22)(q13;q12)) in CCS and EWS-FLI1 (t(11;22)(q24;q12)) in ES. There is a strong premise that targeting the fusion proteins or their key downstream signaling pathways will be disease modifying and improve outcomes for patients. Preliminary high throughput screening studies using an FDA approved small molecule compound library identified filanesib, a kinesin spindle protein (KIF11) inhibitor, as having potent anti-proliferative activity in ES and CCS cells over other cancer subtypes. The mechanism linking the fusion proteins with KIF11 and the tumorigenic role of KIF11 in ES and CCS has yet to be described. Previous studies have shown that the EWS-ATF1 fusion protein forms a heterodimer with transcription factors to target the promoters of its downstream genes. In breast cancer, the knockdown of KIF11 has been shown to significantly reduce the phosphorylation of these transcription factors. Additionally, KIF11 has been shown to directly interact with p300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT). HATs are necessary for EWS-FLI1-induced chromatin remodeling. We hypothesize that the fusion proteins, EWS-FLI1 and EWS-ATF1, form a complex with KIF11 that regulates the activity of co-transcription factors during chromatin remodeling. The target of filanesib, KIF11, has essential roles in maintaining cell cycle progression through mitosis and its inhibition results in mitotic arrest and subsequent cell death. We used ImageStream to assess the effects of filanesib on the cell cycle. ImageStream allows both a quantitative and qualitative analysis of the cell cycle by measuring the population of cells that are positive for a given marker as well as imaging each cell by microscopy. Results demonstrate that filanesib promotes an increase in cells arrested in prophase with dramatically altered spindle formation. We are currently addressing the role of KIF11 in regulating the expression levels of the fusion proteins’ associated transcriptional coactivators and downstream target genes. By completing these studies, we aim to, i) outline the mechanism underpinning the relationship between KIF11 and the oncogenic fusion proteins in ES and CCS cells, and ii) gain an understanding of why ES and CCS cells are particularly vulnerable to KIF11 inhibition. This work has been supported in part by the Flow Cytometry and the Molecular Genomics Cores at the H. Lee Moffitt Cancer Center & Research Institute, a comprehensive cancer center designated by the National Cancer Institute and funded in part by Moffitt’s Cancer Center Support Grant (P30-CA076292) Citation Format: Hannah L. Walker-Mimms, Nicole Londono, Yi Liao, Neelkamal Chaudhary, Dessiree Pena Gomez, Yonghong Zhang, Mingxiang Teng, Uwe Rix, Derek R. Duckett. FDA approved library screen revealed Ewing and clear cell sarcomas have increased sensitivity to filanesib over other cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 647.

HDAC Inhibitors Induce HLA Class I Molecules through the SOX10-IRF1 Axis in Clear Cell Sarcoma Cells.

Although immune checkpoint inhibitors (ICIs) are an effective treatment for clear cell sarcoma (CCS), a rare melanocytic sarcoma with a poor prognosis, their efficacies are still limited. Therefore, a novel therapeutic strategy is required to improve the efficacy of ICIs. We previously reported that histone deacetylase (HDAC) inhibitors increased melanoma immunogenicity through the SOX10-IRF1 pathway and may improve the efficacy of ICIs for melanoma. We herein demonstrated that the inhibition of HDAC induced the expression of HLA class I molecules through IRF1 in CCS cells, similar to melanoma. The suppression of SOX10 by small interfering RNA (siRNA) induced the expression of HLA class I molecules. In addition, the isoform-specific inhibition of HDAC1/3 induced the expression of another IRF1 downstream molecule, PD-L1 in CCS cells in concert with the suppression of SOX10. Furthermore, the knockdown of IRF1 impaired the induction of PD-L1 expression in CCS cells. Therefore, the inhibition of HDAC1/3 has potential as a novel strategy to increase immunogenicity and as combination therapy with ICIs for CCS and melanoma.

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 CCSis found.

Whole‐exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets

Whole‐exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets

An obstructive role of NK cells on metastatic growth of clear-cell sarcoma cells in a xenoplant murine model.

Clear cell sarcoma (CCS) affects the deep soft tissues in young adults and is known to have high rates of metastasis, including lymphatic metastasis. In our previous study an xenoplant model of CCS was established, which exhibited local tumor growth, lymphatic metastasis, and distant metastasis in SCID-Beige mice. In the current study, the role of NK cells during metastasis in the same xenoplant murine model was investigated. Injection of murine or human NK cells significantly suppressed the metastasis of HS-MM CCS cells in SCID-Beige mice. Notably, reverse transcription-quantitative PCR analysis demonstrated that injection of NK cells did not alter the mRNA expression levels of ERSR1-ATF1, which is specifically transcribed in CCS, in the buffy coat of circulating blood cells of HS-MM-xenoplanted SCID-Beige mice. BALB/c nude mice xenoplanted with HS-MM cells exhibited local growth without evident metastasis, whereas inoculation with the anti-asialo-GM1 antibody, which has previously been found to abolish NK-cell activity, resulted in metastasis of HS-MM cells in BALB/c nude mice. The injection of the anti-CD96 antibody, which increases the cytotoxicity of NK cells, significantly suppressed the metastasis of HS-MM cells in SCID-Beige mice. These results indicated that NK cells impaired the metastatic tumor microenvironments in the present mice xenoplant model.

Cell-type dependent enhancer binding of the EWS/ATF1 fusion gene in clear cell sarcomas

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3-expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development.

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.

Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required. A new human CCS cell line (designated RSAR001) was established from the pleural effusion of a 44-year-old man with multiple lung metastases and pleural dissemination. The cell line and its xenograft were characterized including their morphology, immunohistochemistry, cytogenetic analysis, reverse transcription-polymerase chain reaction, direct sequencing analysis, and fluorescence in situ hybridization analysis. The cell line has been maintained for over 12 months with more than 50 passages. RSAR001 cells exhibited a fascicular or diffuse growth pattern of short spindle- or oval-shaped cells with clear cytoplasm in heterotransplanted tumor, that was similar to the primary tumor. Immunophenotypically, RSAR001 cells in vitro and in vivo exhibited almost the same characteristics as the primary tumor. Cytogenetic analyses revealed a translocation, t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction and direct sequencing analysis detected transcripts of the Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) type 1 fusion gene. Fluorescence in situ hybridization using a break-apart probe for the EWSR1 gene on 22q12 showed a rearrangement. These findings indicate that the RSAR001 cell line harbors EWSR1-ATF1 type 1 chimeric fusion gene, which is specific to CCS. RSAR001 cells might be useful for investigating biological behaviors and developing new treatments such as molecular-targeting antitumor drugs or immunological drugs for CCS.

Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma.

Clear cell sarcoma (CCS) is an aggressive type of soft tissue tumor that is associated with high rates of metastasis. In the present study, we found that CPI-613, which targets tumorous mitochondrial energy metabolism, induced autophagosome formation followed by lysosome fusion in HS-MM CCS cells in vitro. Interestingly, CPI-613 along with chloroquine, which inhibits the fusion of autophagosomes with lysosomes, significantly induced necrosis of HS-MM CCS cell growth in vitro. Subsequently, we established a murine orthotropic metastatic model of CCS and evaluated the putative suppressive effect of a combination of CPI-613 and chloroquine on CCS progression. Injection of HS-MM into the aponeuroses of the thigh, the most frequently affected site in CCS, resulted in massive metastasis in SCID-beige mice. By contrast, intraperitoneal administration of CPI-613 (25 mg/kg) and chloroquine (50 mg/kg), two days a week for two weeks, significantly decreased tumor growth at the injection site and abolished metastasis. The present results imply the inhibitory effects of a combination of CPI-613 and chloroquine on the progression of CCS.

Establishment and proteomic characterization of patient-derived clear cell sarcoma xenografts and cell lines.

Clear cell sarcoma (CCS) is an aggressive mesenchymal malignancy characterized by the unique chimeric EWS-ATF1 fusion gene. Patient-derived cancer models are essential tools for the understanding of tumorigenesis and the development of anti-cancer drugs; however, only a limited number of CCS cell lines exist. The objective of this study was to establish patient-derived CCS models. We established patient-derived CCS models from a 43-yr-old female patient. We prepared the patient-derived xenografts (PDXs) from tumor tissues obtained through biopsy or surgery and isolated stable cell lines from PDXs and the original tumor tissue. The presence of gene fusions was examined by RT-PCR, and Sanger sequencing. The established cell lines were characterized by short tandem repeat, viability, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell lines were examined by mass spectrometry and KEGG pathway analysis. The cell lines were maintained for more than 80 passages, and had tumorigenic characteristics such as colony and spheroid formation and invasion. Mass spectrometric proteome analysis demonstrated that the cell lines were enriched for similar but distinct molecular pathways, compared to those in the xenografts and original tumor tissue. Next, tyrosine kinase inhibitors were screened for their suppressive effects on viability. We found that ponatinib, vandetanib, and doxorubicin suppressed the growth of cell lines, and had equivalent IC50 values. Further in-depth investigation and understanding of drug-sensitivity mechanisms will be important for the clinical applications of our cell lines.

Abstract 2141: TAS-115, a novel and highly potent VEGFR/MET inhibitor, shows prominent antitumor efficacy by restoring HGF-mediated hypoxia-resistant phenotype in clear cell sarcoma

Abstract <Background> Hepatocyte growth factor (HGF) / HGF receptor (MET) signaling is considered to be involved in chemoresistance to cancer treatment. Notably, many reports have described reduced sensitivity to vascular endothelial growth factor receptor (VEGFR)-targeted inhibitors in patients with high serum HGF levels compared to those with low serum levels. This study investigated whether HGF directly influenced sensitivity to VEGFR-targeted inhibitors in human clear cell sarcoma (CCS) xenograft models using human HGF knock-in (hHGF KI) mice, and evaluated the potency of TAS-115. <Material and methods> MET signaling pathway in SU-CCS-1, a human CCS cell line, were analyzed using Western blotting analysis. To study the effects of hypoxia in SU-CCS-1 cells, culture plates were placed in airtight jars under anaerobic conditions and cell viability was assessed by ATP-based assay. In the in vivo study, SU-CCS-1 cells were subcutaneously implanted into hHGF KI mice, and compounds were orally administered once daily for 14 consecutive days. Tumor vessel density (TVD) was determined by immunohistochemistry using anti-CD31 antibody. <Results> SU-CCS-1 cells expressed MET, and exogenous HGF phosphorylated MET and its downstream factors in SU-CCS-1 cells. Exogenous HGF also significantly enhanced SU-CCS-1 cell proliferation. TAS-115 inhibited HGF-induced MET phosphorylation and SU-CCS-1 cell proliferation in a dose-dependent manner at concentrations higher than 10 nM. In contrast, pazopanib, a VEGFR-targeted multi-kinase inhibitor, could not block HGF-induced phenotypes in SU-CCS-1 cells, even at 1 μM. To evaluate the effects of HGF on the antitumor activities of TAS-115 and pazopanib against SU-CCS-1 cells, these agents were administered in hHGF KI and wild-type (WT) mice bearing subcutaneous SU-CCS-1 tumors. TAS-115 completely suppressed tumor growth and reduced TVD in SU-CCS-1 tumor tissue in both models at a dose of 200 mg/kg/d. In contrast, pazopanib suppressed tumor growth at a dose of 100 mg/kg/d in WT mice but not in hHGF KI mice. However, pazopanib significantly reduced TVD in SU-CCS-1 tumor tissue in both models. HGF is therefore suggested to impact SU-CCS-1 cells rather than mouse endothelial cells. We considered that HGF modulated cell proliferation or survival in SU-CCS-1 cells under hypoxia. Actually, hypoxic conditions induced apoptosis in SU-CCS-1 cells, and endogenous HGF dose-dependently attenuated hypoxia-related apoptosis. TAS-115 clearly restored HGF-mediated cytoprotection under hypoxia but pazopanib did not. <Conclusion> These results suggest that effects of VEGFR-targeted inhibitors are attenuated by HGF-mediated hypoxia resistance, and simultaneous inhibitors of the MET and VEGFR axes, such as TAS-115, are more effective in sarcoma patients with high serum HGF than inhibition of the VEGFR axis alone. Citation Format: Hidenori Fujita, Yukari Yamada, Miki Terasaka, Yayoi Fujioka, Naomoto Harada, Akihiro Hashimoto, Kazutaka Miyadera, Kenichi Matsuo, Kazuhiko Yonekura. TAS-115, a novel and highly potent VEGFR/MET inhibitor, shows prominent antitumor efficacy by restoring HGF-mediated hypoxia-resistant phenotype in clear cell sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2141.

Abstract 3777: TAS4464, a novel and highly potent NEDD8 activating enzyme (NAE) inhibitor, causes apoptosis of sarcomas via cell cycle dysregulation

Abstract Background Soft-tissue sarcomas (STSs) are heterogeneous tumors that comprise approximately 1% of adult cancers. Patients with advanced STS have a poor prognosis because few chemotherapy options are available. NAE catalyzes the first step in the NEDD8 conjugation (neddylation) pathway. Because it activates cullin-RING ligase complexes (CRLs) and thus is essential in cancer cell homeostasis, NAE is a promising target for cancer therapy. Here, we investigated the potency of the NAE inhibitor TAS4464 for various STS cell lines. Material and methods Cytotoxicity was evaluated through ATP-Based assay. The effects of TAS4464 on NEDD8 conjugation and CRL substrates were evaluated by Western analysis. Cell cycle progression was analyzed by using flow cytometry. Small interfering RNAs (siRNAs) were lipofected into a clear cell sarcoma (CCS) line (SU-CCS-1). The antitumor activity of intravenous TAS4464 was evaluated in xenograft models of the aforementioned CCS and 2 rhadomyosarcoma (RMS) lines (SJCRH30 and RD). Results TAS4464 suppressed cell growth and induced cell death in various STS cell lines at lower concentrations than did an investigational NAE inhibitor, MLN4924, and a conventional STS treatment agent, doxorubicin (DXR). Notably, the GI50 values of TAS4464 were less than 10 nM in the tested RMS and CCS cell lines. TAS4464 treatment led to the elimination of cullin neddylation, accumulation of CRL substrate proteins (CDT1, p27, and p21), S phase arrest, and ultimately apoptosis in these STS cell lines. Furthermore, knockdown of CRL substrate proteins by siRNAs markedly attenuated TAS4464-induced cytotoxicity in STS cells. These results suggest that TAS4464-induced cell death was triggered by cell cycle dysregulation based on accumulation of CRL substrate proteins following NAE inhibition. TAS4464 also led to a decrease in cullin neddylation and accumulation of CRL substrate proteins in RMS and CCS lines subcutaneous xenografts. Weekly administration of TAS4464 (100 mg/kg, IV) completely suppressed tumor growth in mice bearing subcutaneous RD and SJCRH30 xenografts. The effects of TAS4464 significantly exceeded that of DXR and MLN4924. Furthermore, TAS4464 induced tumor regression of ∼50% in the pazopanib, a drug approved for STS, insensitive SU-CCS-1 xenograft model. Conclusion TAS4464 prominently inhibited cell growth and induced apoptosis in RMS and CSC cells through cell cycle dysregulation and demonstrated impressive antitumor activities in STS xenograft models that respond poorly to DXR and pazopanib. Therefore, TAS4464 may become a valuable therapeutic option for patients with advanced STS. Citation Format: Hidenori Fujita, Yayoi Fujioka, Keiji Ishida, Chihoko Yoshimura, Akihiro Hashimoto, Shingo Tsuji, Takashi Mizutani, Shuichi Okubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel and highly potent NEDD8 activating enzyme (NAE) inhibitor, causes apoptosis of sarcomas via cell cycle dysregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3777.

Boron neutron capture therapy as new treatment for clear cell sarcoma: Trial on different animal model

Clear cell sarcoma (CCS) is a rare malignant tumor with a poor prognosis. In our previous study, the tumor disappeared under boron neutron capture therapy (BNCT) on subcutaneously-transplanted CCS-bearing animals. In the present study, the tumor disappeared under this therapy on model mice intramuscularly implanted with three different human CCS cells. BNCT led to the suppression of tumor-growth in each of the different model mice, suggesting its potentiality as an alternative to, or integrative option for, the treatment of CCS.

Boron neutron capture therapy for clear cell sarcoma (CCS): Biodistribution study of p-borono-l-phenylalanine in CCS-bearing animal models

Clear cell sarcoma (CCS) is a rare melanocytic malignant tumor with a poor prognosis. Our previous study demonstrated that in vitro cultured CCS cells have the ability to highly uptake l-BPA and thus boron neutron capture therapy could be a new option for CCS treatment. This paper proved that a remarkably high accumulation of (10)B (45-74 ppm) in tumor was obtained even in a CCS-bearing animal with a well-controlled biodistribution followed by intravenous administration of L-BPA-fructose complex (500 mg BPA/kg).

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma.

Clear cell sarcoma (CCS) is associated with the EWS/ATF1 oncogene that is created by chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. The melanocytic character of CCS suggests that the microphthalmia-associated transcription factor (Mitf), a major inducer of melanocytic differentiation, may be miss-expressed in CCS. Accordingly, we show that the mRNA and protein of the melanocyte-specific isoform of Mitf (Mitf-M) are present in several cultured CCS cell lines (Su-ccs-1, DTC1, Kao, MST-1, MST-2 and MST-3). The above cell lines thus provide a valuable experimental resource for examining the role of Mitf-M in both CCS and melanocyte differentiation. Melanocyte-specific expression of Mitf-M is achieved via an ATF-dependent melanocyte-specific cAMP-response element in the Mitf-M promoter, and expression of Mitf-M in CCS cells suggests that EWS/ATF1 (a potent and promiscuous activator of cAMP-inducible promoters) may activate the Mitf-M promoter. Surprisingly, however, the Mitf-M promoter is not activated by EWS/ATF1 in transient assays employing CCS cells, melanocytes or nonmelanocytic cells. Thus, our results indicate that Mitf-M promoter activation may require an appropriate chromosomal context in CCS cells or alternatively that the Mitf-M promoter is not directly activated by EWS/ATF1.

Newly established clear cell sarcoma (malignant melanoma of soft parts) cell line expressing melanoma-associated Melan-A antigen and overexpressing C-MYC oncogene

Clear cell sarcoma (CCS), malignant melanoma of soft parts, is a rare malignant tumor with a poor prognosis. In this study, a CCS cell line, designated MP-CCS-SY, was established from a metastatic tumor of a 17-year-old Japanese girl that originated in the left Achilles tendon. A small number of melanosomes were detected in the cytoplasm by electron microscopy. The melanosomes immunoreacted with two melanoma-associated antibodies, HMB45 and Melan-A. A Western blot demonstrated the existence of a Melan-A antigen in this cell line. Although a t(12;22)(q13;q12), which is characteristic of CCS, was not identified by a chromosomal analysis with conventional banding techniques, fluorescence in situ hybridization analysis with painting probes of chromosomes 12 and 22 revealed the insertion of a chromosome 12 fragment into one of the long arms of chromosome 22. The chimeric EWS/ATF1 transcript was detected by the reverse transcriptase polymerase chain reaction. Extra copies and structural abnormalities of chromosome 8 were observed. Overexpression of c-myc mRNA was detected by Northern blot analysis and may have a role in malignant progression of CCS. The availability of this MP-CCS-SY cell line will help to understand the molecular biology of this malignancy and should be useful as a tool for developing an immunotherapy.