Desmoplastic Small Round Cell Tumor Cells Research Articles

Enzalutamide induces cytotoxicity in desmoplastic small round cell tumor independent of the androgen receptor

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, pediatric cancer caused by the EWSR1::WT1 fusion protein. DSRCT predominantly occurs in males, which comprise 80-90% of the patient population. While the reason for this male predominance remains unknown, one hypothesis is that the androgen receptor (AR) plays a critical role in DSRCT and elevated testosterone levels in males help drive tumor growth. Here, we demonstrate that AR is highly expressed in DSRCT relative to other fusion-driven sarcomas and that the AR antagonists enzalutamide and flutamide reduce DSRCT growth. However, despite these findings, which suggest an important role for AR in DSRCT, we show that DSRCT cell lines form xenografts in female mice at the same rate as male mice and AR depletion does not significantly alter DSRCT growth in vitro. Further, we find that AR antagonists reduce DSRCT growth in cells depleted of AR, establishing an AR-independent mechanism of action. These findings suggest that AR dependence is not the reason for male predominance in DSRCT and that AR-targeted therapies may provide therapeutic benefit primarily through an AR-independent mechanism that requires further elucidation.

Abstract 6: Defining and therapeutically targeting a fusion-derived public neoantigen in desmoplastic small round cell tumor using T cell receptor gene therapy

Abstract Sarcomas are a heterogenous group of mesenchymal cell malignancies that are difficult to treat and often respond poorly to current immunotherapies. Approximately 30% of sarcomas are associated with oncogenic driver fusion proteins. Desmoplastic small round cell tumor (DSRCT) is a prototypical fusion-driven sarcoma defined by a pathognomonic EWSR1-WT1 fusion event. The resultant EWSR1-WT1 fusion protein contains a shared junctional amino acid sequence that is clonally conserved and significantly divergent from normal human proteins. Consequently, the EWSR1-WT1 fusion might yield a highly immunogenic shared, or public, neoantigen (NeoAg) that may serve as a target for novel immunotherapeutic approaches.Using a functional HLA-immunoprecipitation/mass spectrometry (HLA-IP/MS) screen, we identified a 9-amino acid peptide sequence derived from the junction of the most common EWSR1-WT1 fusion protein that is presented in the context of the prevalent HLA-A*03 and -A*11 alleles. We confirmed that HLA-A*03+ DSRCT cells physiologically present the same peptide sequence. Using fluorophore-conjugated HLA-multimers loaded with the HLA-IP/MS-derived fusion NeoAg, we identified a rare population of circulating NeoAg-specific CD8+ T cells in HLA+ DSRCT patients. Through antigen-directed clonal expansion, we discovered n=3 HLA-A*03-restricted and n=1 HLA-A*11-restricted fusion NeoAg-specific T cell clones and retrieved the paired TCRαβ gene sequences of their T cell receptors (TCRs) using 10x single-cell sequencing. Polyclonal CD8+ T cells transduced with candidate TCRαβ gene sequences robustly upregulated TNFα after coculture with HLA+/Fusion+ target cells but not HLA−/Fusion+ or HLA+/Fusion− control cells. Further, T cells expressing candidate TCRs, but not viral-protein-specific TCRs, lysed HLA+ DSRCT cells in vitro and controlled established DSRCT tumors in vivo. Finally, we discovered that one TCR was capable of cross HLA allele fusion NeoAg recognition and could lyse both HLA-A*03+ and -A*11+ DSRCT cells. These findings imply that a single TCR therapeutic could cover >36% of North American DSRCT patients.Collectively, our data establish for the first time that DSRCT cells present a junction-spanning fusion-derived neoantigen in the context of two prevalent HLA alleles that may be therapeutically targeted using TCR gene therapy. These data lay the foundation for first-in-human clinical translation of a T-cell based therapy targeting EWSR1-WT1. More broadly, these findings establish proof-of-principle that recurrent fusion proteins are an actionable source of immunogenic public NeoAgs, providing a framework for developing targeted immunotherapies for other fusion-driven malignancies. Citation Format: Lauren Banks, Hannah Arkin, Smita Chandran, Emily Slotkin, Madelyn Espinosa-Cotton, Neerav Shukla, Luc Morris, Marc Ladanyi, Andrew Kung, Martin Klatt, Christopher Klebanoff. Defining and therapeutically targeting a fusion-derived public neoantigen in desmoplastic small round cell tumor using T cell receptor gene therapy [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 6.

Abstract 6932: Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in desmoplastic small round cell tumors

Abstract Background: Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, aggressive sarcoma primarily affecting young males. Despite being driven by the unique oncogenic transcription factor (TF) EWSR1-WT1, and a monotonous morphology, DSRCT cells display a polyphenotypic differentiation of unknown etiology. Methods: To explore DSRCT heterogeneity, we characterized a series of patient samples by single-cell, spatial, and bulk multi-omics. We integrated patients’ data with bulk RNA-sequencing, ChIP-sequencing, and ATAC-sequencing data generated in a DSRCT cell line model upon EWSR1-WT1 transcriptional silencing. Results: Single-cell RNA-sequencing (scRNA-seq) on 11 patient samples identified recurrent subpopulations of DSRCT tumor cells, including lineage-, metabolism-, and pseudostate-related clusters. These subpopulations showed overlapping gene modules and lacked copy number variation diversity, suggesting transcriptional plasticity as a potential source of tumor heterogeneity. We first investigated whether cell-intrinsic mechanisms may drive DSRCT intra-tumor heterogeneity. Using an in-house-developed assay, we found limited variability in EWSR1-WT1 transcript expression. Preclinical modeling defined an EWSR1-WT1 regulon, used as a surrogate for EWSR1-WT1 TF activity. Integration with scRNA-seq data showed that high EWSR1-WT1 activity was associated with lineage-related states, while glycolytic and profibrotic states correlated with low EWSR1-WT1 activity. Single-cell ATAC-seq with scRNA-seq (Multiome) on one patient sample identified TF motifs related to developmental processes - e.g., SOX, FOX, and GATA - as the most differentially enriched across clusters. Interestingly, EGR1 regulon activity showed an inverse correlation with EWSR1-WT1 and Androgen Receptor regulons’ activity, in line with preclinical data, where ATAC-seq showed increased accessibility of EGR1 upon EWSR1-WT1 silencing. We next investigated cell-extrinsic factors by spatial transcriptomics in six patient samples. Glycolytic and profibrotic subpopulations localized within hypoxic niches at the tumor periphery, in direct proximity to cancer-associated fibroblasts (CAFs), which promoted tumor growth in vitro, as assessed in DSRCT cells/CAFs co-culture assays. Finally focusing on inter-tumor heterogeneity, the prognostic evaluation of scRNA-seq-derived signatures revealed that a tumor cell epithelial signature correlated with improved patient survival in a cohort of 29 DSRCT samples profiled with bulk RNA-sequencing. Conclusion: DSRCT heterogeneity is driven by combined cell-intrinsic and -extrinsic mechanisms, including EWSR1-WT1 DNA-binding activity and interaction with CAFs. Gene signatures associated with specific DSRCT subpopulations correlated with prognosis, highlighting the clinical relevance of our findings. Citation Format: Clémence Henon, Julien Vibert, Thomas Eychenne, Nadège Gruel, Léo Colmet-Daage, Carine Ngo, Marlène Garrido, Nicolas Dorvault, Maria Eugenia Marques Da Costa, Antonin Marchais, Asuka Kawachi, Madison Lenormand, Clémence Astier, Roman Chabanon, Benjamin Verret, Rastislav Bahleda, Axel Le Cesne, Fatima Mechta-Grigoriou, Charles Honoré, Matthieu Faron, Olivier Delattre, Joshua J Waterfall, Sarah Watson, Sophie Postel-Vinay. Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in desmoplastic small round cell tumors [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 6932.

Transcriptomic analysis identifies B-lymphocyte kinase as a therapeutic target for desmoplastic small round cell tumor cancer stem cell-like cells

Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. The tumor is refractory to treatment with a 5-year survival rate of only 15–25%, necessitating the development of novel therapeutics, especially those able to target chemoresistant subpopulations. Novel in vitro cancer stem cell-like (CSC-like) culture conditions increase the expression of stemness markers (SOX2, NANOG) and reduce DSRCT cell line susceptibility to chemotherapy while maintaining the ability of DSRCT cells to form xenografts. To gain insights into this chemoresistant model, RNA-seq was performed to elucidate transcriptional alterations between DSRCT cells grown in CSC-like spheres and normal 2-dimensional adherent state. Commonly upregulated and downregulated genes were identified and utilized in pathway analysis revealing upregulation of pathways related to chromatin assembly and disassembly and downregulation of pathways including cell junction assembly and extracellular matrix organization. Alterations in chromatin assembly suggest a role for epigenetics in the DSRCT CSC-like state, which was further investigated with ATAC-seq, identifying over 10,000 differentially accessible peaks, including 4444 sphere accessible peaks and 6,120 adherent accessible peaks. Accessible regions were associated with higher gene expression, including increased accessibility of the CSC marker SOX2 in CSC-like culture conditions. These analyses were further utilized to identify potential CSC therapeutic targets, leading to the identification of B-lymphocyte kinase (BLK) as a CSC-enriched, EWSR1-WT1-regulated, druggable target. BLK inhibition and knockdown reduced CSC-like properties, including abrogation of tumorsphere formation and stemness marker expression. Importantly, BLK knockdown reduced DSRCT CSC-like cell chemoresistance, making its inhibition a promising target for future combination therapy.

Identification of immunotherapy and radioimmunotherapy targets on desmoplastic small round cell tumors.

Development of successful antibody-based immunotherapeutic and radioimmunotherapeutic strategies rely on the identification of cell surface tumor-associated antigens (TAA) with restricted expression on normal tissues. Desmoplastic small round cell tumor (DSRCT) is a rare and generally neglected malignancy that primarily affects adolescent and young adult males. New therapies capable of treating disseminated disease are needed for DSRCT, which is often widespread at diagnosis. We used immunohistochemistry (IHC) on fresh frozen surgical specimens and patient-derived xenograft (PDX) tumors and flow cytometry on DSRCT cell lines to evaluate expression of TAAs in these tumors. In vitro cytotoxicity assays were used to evaluate the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets. In vivo, we used an intraperitoneal xenograft mouse model of DSRCT to test T-BsAbs against several TAAs. In DSRCT specimens we found widespread expression of B7-H3, EGFR, GD2, HER2, mesothelin, and polysialic acid, clinical targets for which specific antibody therapeutics are available. The expression of B7-H3, EGFR, HER2, and mesothelin was confirmed on the cell surface of DSRCT cell lines. In vitro cytotoxicity assays confirmed the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets against DSRCT cells. Remarkably, a HER2xCD3 T-BsAb was capable of completely shrinking established tumors in an intraperitoneal mouse model of DSRCT. We propose that these TAAs should be further investigated in preclinical models as targets for immunotherapy and radioimmunotherapy with the hope of providing a rationale to extend these therapies to patients with advanced DSRCT.

Abstract 6277: Identification of ATR inhibitors as therapeutic opportunities in Desmoplastic small round cell tumors

Abstract Introduction: Desmoplastic small round cell tumor (DSRCT) is a rare and very aggressive subtype of sarcoma, affecting predominantly young males. DSRCT is molecularly characterized by the t(11;22)(q24;q12) chromosomal translocation, which fuses EWSR1 to WT1 and encodes an aberrant chimeric transcription factor. A few additional secondary mutations have been reported, notably in genes encoding proteins involved in chromatin remodeling and DNA repair. No therapeutic advance have been done in the past 20 years, and the five-year overall survival is still below 15% in metastatic cases, despite aggressive poly-chemotherapy and extensive surgical debulking. Thus, we aimed to identify actionable targeted dependencies to propose novel therapeutic approaches, using drug screening and molecular biology. Material and Methods: Drug sensitivity screen evaluating 80 cancer-relevant small molecule inhibitors in dose-response was performed on the JN-DSRCT-1 (JN1) cell line. Revalidation was performed on JN1 and in a Champions Oncology PDX-derived in-house-generated DSRCT cell line (R) using multiple clinical compounds. Mechanistic dissection was performed using flow cytometry, immunofluorescence, western blotting, DNA fiber assay, RT-qPCR, and dot blot to evaluate markers of DNA damage response, replication stress and cell-autonomous innate immune signaling. Results: The drug screen identified that JN1 cells were particularly sensitive to one ATR inhibitor (ATRi) and multiple PARPi inhibitors (PARPi), which scored #6, 7, 9 and 20 of the hits. Sensitivity to ATRi was revalidated in the JN1 and R cell lines, whereas sensitivity to PARPi was only revalidated in the JN1 cell line - consistent with the very low PARP1 expression in that cell line. Sensitivity to ATRi and PARPi was partially reversed upon EWS-WT1 silencing. Combination experiments revealed that both drugs were synergistic in the JN1 cell line. Exposure to PARP inhibitor increased the number of γH2AX and RAD51 foci, suggesting that JN1 cells were not deficient in homologous recombination. Further, PARPi or ATRi exposure decreased replication fork speed and increased R-loops accumulation, whereas EWS-WT1 silencing increased replication fork speed. Resistance to PARPi and to ATRi was restored upon exogenous expression of the R-loop resolution enzyme RNAseH1. The combination of PARPi and ATRi also induced micronuclei formation and activated the cGAS-STING cytosolic innate immune sensing pathway, together with increased expression of CCL5, CXCL10 and PD-L1. Conclusion: DSRCT cells are sensitive to ATR and PARP inhibitors, through a mechanism that, at least in part, may involve enhanced replication stress and increased R-loop formation. Our data provide the preclinical rationale for assessing ATRi combined with PARPi in patients with DSRCT, as a cytotoxic therapy or as an immunomodulatory approach to enhance cell-autonomous immunogenicity. Citation Format: Asuka KAWAI-KAWACHI, Madison LENORMAND, Clémence HENON, Thomas EYCHENNE, Leo COLMET-DAAGE, Nicolas DORVAULT, Marlene GARRIDO, Clémence ASTIER, Carine NGO, Helen PEMBERTON, Aditi GULATI, Stephen PETTITT, Roman CHABANON, Christopher J LORD, Sophie POSTEL-VINAY. Identification of ATR inhibitors as therapeutic opportunities in Desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6277.

The androgen receptor is a therapeutic target in desmoplastic small round cell sarcoma

Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5α-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have direct clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer.

Salt-Inducible Kinase 1 is a potential therapeutic target in Desmoplastic Small Round Cell Tumor

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by a chromosomal translocation t(11;22)(p13;q12) that produces an oncogenic transcription factor, EWSR1-WT1. EWSR1-WT1 is essential for the initiation and progression of DSRCT. However, the precise mechanism by which EWSR1-WT1 drives DSRCT oncogenesis remains unresolved. Through our integrative gene expression analysis, we identified Salt Inducible Kinase 1 (SIK1) as a direct target of EWSR1-WT1. SIK1 as a member of the AMPK related kinase is involved in many biological processes. We showed that depletion of SIK1 causes inhibition of tumor cell growth, similar to the growth inhibition observed when EWSR1-WT1 is depleted. We further showed that silencing SIK1 leads to cessation of DNA replication in DSRCT cells and inhibition of tumor growth in vivo. Lastly, combined inhibition of SIK1 and CHEK1with small molecule inhibitors, YKL-05-099 and prexasertib, respectively, showed enhanced cytotoxicity in DSRCT cells compared to inhibition of either kinases alone. This work identified SIK1 as a new potential therapeutic target in DSRCT and the efficacy of SIK1 inhibition may be improved when combined with other intervention strategies.

EWSR1-WT1 Target Genes and Therapeutic Options Identified in a Novel DSRCT In Vitro Model

Simple SummaryDesmoplastic small round cell tumor (DSRCT) is an extremely rare soft tissue sarcoma arising in the abdomen of adolescents and young adults. This sarcoma is driven by a single genomic rearrangement, resulting in the expression of the EWSR1-WT1 fusion gene. No effective treatment exists for DSRCT patients, which highlights the need for preclinical models to study disease progression and drug sensitivity. The aim of this study is to develop a pre-clinical DSRCT in vitro model, which enables investigating the molecular target genes of the EWSR1-WT1 fusion gene and allows for medium-throughput drug screening to discover new therapeutic options.Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft tissue sarcoma with a lack of effective treatment options and a poor prognosis. DSRCT is characterized by a chromosomal translocation, resulting in the EWSR1-WT1 gene fusion. The molecular mechanisms driving DSRCT are poorly understood, and a paucity of preclinical models hampers DSRCT research. Here, we establish a novel primary patient-derived DSRCT in vitro model, recapitulating the original tumor. We find that EWSR1-WT1 expression affects cell shape and cell survival, and we identify downstream target genes of the EWSR1-WT1 fusion. Additionally, this preclinical in vitro model allows for medium-throughput drug screening. We discover sensitivity to several drugs, including compounds targeting RTKs. MERTK, which has been described as a therapeutic target for several malignancies, correlates with EWSR1-WT1 expression. Inhibition of MERTK with the small-molecule inhibitor UNC2025 results in reduced proliferation of DSRCT cells in vitro, suggesting MERTK as a therapeutic target in DSRCT. This study underscores the usefulness of preclinical in vitro models for studying molecular mechanisms and potential therapeutic options.

Trabectedin (T) in desmoplastic small round cell tumor (DSRCT): Report of its effect in 3 relapsed patients (pts) and the comparison of different regimens in a patient-derived xenograft (PDX) model.

e23553 Background: DSRCT is an ultra-rare soft tissue sarcoma marked by the presence of the EWS-WT1 translocation and a dismal prognosis. Anecdotal activity of T in DSRCT pts was reported. We describe herein three advanced DSRCT pts treated with T and a comparative assessment of doxorubicin (D), pazopanib (P) and T in a patient-derived xenograft (PDX) model of DSRCT. Methods: Three pts (#1, #2 and #3) suffering from progressive, metastatic, unresectable relapsing disease from a primary peritoneal DSRCT previously treated with 8 cycles of anthracycline-based neoadjuvant chemo and complete surgical resection, were started on T (1.3 mcg/sqm every 3-4 weeks). A PDX model was generated by subcutaneously implanting small tumor fragments obtained at surgery from a treatment-naïve DSRCT patient into the right flank of SCID mice. Consistency of PDX and the originating tumor was confirmed in terms of histomorphology and presence of the EWS-WT1 gene fusion. Mice were randomized to receive D, P and T, administered as single agents at optimal doses and schedules. Drug activity was assessed in terms of tumor volume inhibition (TVI) percentage in treated versus control mice. An orthotopic xenograft model was also generated by injecting DSRCT cells into the peritoneal cavity of SCID mice. Results: At the time of this report, pt #1 and #2 are on therapy with T, with a partial response by RECIST maintained after 48 and 36 months from treatment start, respectively, while #3 progressed after 4 months. In the DSRCT PDX model, T was the most effective drug, with a maximum TVI of 82%, while D and P showed lower, comparable activity (maximum TVI: 59% and 66%, respectively). In the orthotopic DSRCT PDX, DSRCT cells spreading in the abdominal cavity generated different tumor masses, properly recapitulating the dissemination pattern in patients, confirming the reliability of this preclinical model. Conclusions: Both our preliminary model and our further clinical observations support the potential of T in DSRCT. A confirmatory prospective clinical study is now warranted.

Therapeutic Potential of NTRK3 Inhibition in Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a highly lethal intra-abdominal sarcoma of adolescents and young adults. DSRCT harbors a t(11;22)(p13:q12) that generates the EWSR1-WT1 chimeric transcription factor, the key oncogenic driver of DSRCT. EWSR1-WT1 rewires global gene expression networks and activates aberrant expression of targets that together mediate oncogenesis. EWSR1-WT1 also activates a neural gene expression program. Among these neural markers, we found prominent expression of neurotrophic tyrosine kinase receptor 3 (NTRK3), a druggable receptor tyrosine kinase. We investigated the regulation of NTRK3 by EWSR1-WT1 and its potential as a therapeutic target in vitro and in vivo, the latter using novel patient-derived models of DSRCT. We found that EWSR1-WT1 binds upstream of NTRK3 and activates its transcription. NTRK3 mRNA is highly expressed in DSRCT compared with other major chimeric transcription factor-driven sarcomas and most DSRCTs are strongly immunoreactive for NTRK3 protein. Remarkably, expression of NTRK3 kinase domain mRNA in DSRCT is also higher than in cancers with NTRK3 fusions. Abrogation of NTRK3 expression by RNAi silencing reduces growth of DSRCT cells and pharmacologic targeting of NTRK3 with entrectinib is effective in both in vitro and in vivo models of DSRCT. Our results indicate that EWSR1-WT1 directly activates NTRK3 expression in DSRCT cells, which are dependent on its expression and activity for growth. Pharmacologic inhibition of NTRK3 by entrectinib significantly reduces growth of DSRCT cells both in vitro and in vivo, providing a rationale for clinical evaluation of NTRK3 as a therapeutic target in DSRCT.

Mini-Review on Targeted Treatment of Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a devastating disease which most commonly affects adolescents, with a male predominance. Despite the best multimodality treatment efforts, most patients will ultimately not survive more than 3–5 years after diagnosis. Some research trials in soft-tissue sarcoma and Ewing sarcoma include DSRCT patients but few studies have been tailored to the specific clinical needs and underlying cytogenetic abnormalities characterizing this disease such as the typical EWSR1-WT1 gene fusion. Downstream activation of EWSR1-WT1 gene fusion includes signaling pathways of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin growth factor (IGF)-1. Other biological pathways that are activated and expressed in DSRCT cells include endothelial growth factor receptor (EGFR), androgen receptor pathway, c-KIT, MET, and transforming growth factor (TGF) beta. Investigation of somatic mutations, copy number alterations (CNA), and chromosomes in DSRCT samples suggests that deregulation of mesenchymal-epithelial reverse transition (MErT)/epithelial-mesenchymal transition (EMT) and DNA damage repair (DDR) may be important in DSRCT. This mini review looks at known druggable targets in DSRCT and existing clinical evidence for targeted treatments, particularly multityrosine kinase inhibitors such as pazopanib, imatinib, and sorafenib alone or in combination with other agents such as mTOR (mammalian target of rapamycin) inhibitors. The aim is to increase shared knowledge about current available treatments and identify gaps in research to further efforts toward clinical development of targeted agents.

Efficacy of ONC201 in Desmoplastic Small Round Cell Tumor

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare sarcoma tumor of adolescence and young adulthood, which harbors a recurrent chromosomal translocation between the Ewing’s sarcoma gene (EWSR1) and the Wilms’ tumor suppressor gene (WT1). Patients usually develop multiple abdominal tumors with liver and lymph node metastasis developing later. Survival is poor using a multimodal therapy that includes chemotherapy, radiation and surgical resection, new therapies are needed for better management of DSRCT. Triggering cell apoptosis is the scientific rationale of many cancer therapies. Here, we characterized for the first time the expression of pro-apoptotic receptors, tumor necrosis-related apoptosis-inducing ligand receptors (TRAILR1-4) within an established human DSRCT cell line and clinical samples. The molecular induction of TRAIL-mediated apoptosis using agonistic small molecule, ONC201 in vitro cell-based proliferation assay and in vivo novel orthotopic xenograft animal models of DSRCT, was able to inhibit cell proliferation that was associated with caspase activation, and tumor growth, indicating that a cell-based delivery of an apoptosis-inducing factor could be relevant therapeutic agent to control DSRCT.

PARP inhibitor combination therapy in desmoplastic small round cell tumors.

e23212 Background: Desmoplastic small round cell tumor (DSRCT) is a rare but pernicious mesenchymal neoplasm that expresses the EWS-WT1 gene fusion transcript. It has similar histopathology to Ewing sarcoma (EWS). In previous work, we showed that addition of Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi) to the existing standard of care (SOC) therapy of irinotecan (IRN) and temozolomide (TMZ) was remarkably successful at treating mouse models of EWS, resulting in > 80% complete response compared to 100% mortality with SOC alone. EWS has high levels of the protein Schlafen-11 (SLFN11), a putative biomarker for defective DNA damage repair. The goal of this study is to determine whether DSCRT also expresses SLFN11, and whether high levels of the protein sensitize the tumor to PARPi as single-agent or in combination with other drugs and ionizing radiation. Methods: SLFN11 mRNA and protein levels were assessed by immunohistochemistry, quantitative PCR, Western blot, and immunofluorescence microscopy. Viability of the DSRCT cell line, JN-DSCRT-1, was measured using CellTiterGlo and the colony forming assay. Ionizing radiation (IR) was delivered using an orthovoltage irradiator calibrated to deliver graded doses of 0-16 Gy. Results: We found that SLFN11 was detected at high levels in several DSRCT patient tumor samples, and we confirmed that SLFN11 transcript and protein levels in JN-DSCRT-1were comparable to EWS cell lines. JN-DSCRT-1 demonstrated sensitivity to PARPi as single-agent or in combination with either the topoisomerase I inhibitor irinotecan or ionizing radiation comparable to EWS cell lines. To supplement our in vitro studies and to utilize more accurate models of the clinical disease, we developed orthotopic patient derived xenograft models (O-PDX) from three patients with DSCRT. Preliminary findings suggest that these O-PDX models maintain SLFN11 expression and recapitulate important aspects of DSRCT biology, including widespread dissemination of tumor in the abdominal cavity and characteristic cellular phenotype. Preclinical in vivo efficacy studies with PARPi combinations are in progress. Conclusions: Our work suggests that DSCRT expresses the SLFN11 biomarker and is sensitive to PARPi combination therapy.

Abstract LB-238: Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas

Abstract Many sarcoma subtypes including Ewing sarcoma (ES), alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT), are driven by oncogenic chimeric transcription factors. Outcomes for these diseases remain unacceptably poor despite intensification of standard therapies. Although these translocation positive sarcomas possess well-defined oncogenic drivers, as transcription factors that lack intrinsic enzymatic activity, they have been considered undruggable. We have developed a two-tiered pre-clinical therapeutic program 1) creating and characterizing faithful in vitro and in vivo models of these rare malignancies and 2) designing next generation antisense oligonucleotides (ASOs) to directly target the oncogenic chromosomal translocations underlying these multiple sarcoma subtypes. As proof of principle, we present here the program experience with DSRCT, a rare sarcoma for which there has been an overwhelming paucity of both biological tools for pre-clinical investigation, as well as clinical therapeutic options. We have extensively characterized two new DSRCT cell lines harboring the characteristic oncogenic EWSR1-WT1 t(11;22)(p13;q12) fusion, and have used these tools to develop orthotopic xenograft models. Utilizing these accurate model systems we have designed and evaluated a series of ASO compounds directly targeting the EWSR1-WT1 translocation product. Among the series of ASOs developed, we identified D2A, a selective and potent inhibitor of EWSR1-WT1. In vitro, we have shown D2A to abolish the EWSR1-WT1 fusion product with exquisite specificity in a dose-dependent fashion. D2A similarly exhibits efficacy in vivo, with anti-tumorigenic activity in our pre-clinical models resulting in an 80% reduction in xenograft tumor growth. With the aid of authentic in vitro and in vivo modeling systems, this ASO platform has wide-ranging implications for both biological as well as therapeutic advances. Citation Format: Emily Slotkin, Lee Spraggon, Julija Hmeljak, Luciano Martelotto, Romel Somwar, Elisa de Stanchina, Luca Cartegni, Marc Ladanyi. Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas. [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 LB-238.

Abstract 1625: Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues

Abstract Ureidomustine (BO-1055) is a water-soluble N- mustard derivative that has antitumor activity against a number of human tumors including prostate, colon cancers and gliomas. In this study, we investigated its pre-clinical activity in sarcoma models. We tested the activity of BO-1055 on early passages from Ewing sarcoma patient samples and patient-derived xenografts, preclinical models of rhabdomyosarcoma, desmoplastic small round cell tumor (DSRCT) and osteosarcoma. We used cell proliferation, viability, clonogenicity and cell death assays, and immunoblotting for differential expression of proteins in cells that are sensitive and resistant to BO-1055. We studied the toxicity of BO-1055 on cardiac myocytes, hematopoietic stem and progenitor cells (HSPCs), epithelial, mesenchymal and endothelial cells, and performed animal toxicity experiments in mice. Efficacy of BO-1055 was tested in NSG mice bearing A673 xenografts (Ewing sarcoma) treated at doses of 10mg/kg, 20mg/kg and 30mg/kg, Q2D×5, via i.v. injection after the tumors grew to 250-500 mm3. We also investigated the efficacy of this agent in NSG mice bearing A204 xenografts and Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. The antitumor effects of BO-1055 in combination with standard chemotherapeutic agents were also evaluated. Ureidomustine exhibited significant cytotoxicity against the in vitro growth of Ewing sarcoma and rhabdomyosarcoma with sub μM IC50 values and was more cytotoxic than conventional DNA damaging agents. BO-1055 had less cytotoxicity against a panel of normal HSPCs. BO-1055 had moderate activity (IC50 = 2-4 μM) on DSRCT cells and had only weak activity (IC50>10μM) against osteosarcoma cell lines. Notably, this agent has no cross-resistance to Taxol and Vinblastine. Growth arrest in G2/M phase was noted at 24h and maximal apoptosis and cell death at 48h and 72h after exposure of A673 cells. Apoptosis was due to the induction of caspase 3 and 7 activity in a dose- and time- dependent manner. Cardiotoxicity was evaluated by binding of BO-1055 to hERG, which was significantly less than that of Astemizole indicating that BO-1055 has less propensity for QTc prolongation. BO-1055 significantly suppressed Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. Remarkably, this agent induced complete suppression of tumors in nude mice bearing A204 xenografts, without significant weight loss. In two-drug combination studies, BO-1055 exhibited synergism with etoposide, SN-38 (active metabolite of irinotecan), doxorubicin and PU-H71 (HSP-90 inhibitor). Because of its broad therapeutic window, lack of cross-resistance, better safety profile and synergistic cytotoxicity with standard drugs, BO-1055 has high potential for clinical application for treatment of Ewing sarcoma and rhabdomyosarcoma patients. Citation Format: Srikanth R. Ambati, Shieh JaeHung, Benet Pera, Elissa W.P. Wong, Eloisi Caldas Lopes, Elizabeth Peguero, Tsann-Long Su, Malcolm A.S. Moore. Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2015-1625

Desmoplastic Small Round Cell Tumor, a "Floating Island" Pattern in Pleural Fluid Cytology: A Case Report and Review of the Literature.

Desmoplastic small round cell tumor (DSRCT) is a rare aggressive sarcoma with characteristic clinical and pathologic features. It typically involves pelvic and abdominal organs of young male patients, and patients usually present at advanced stage with poor prognosis. A few reports are available describing the cytopathologic features of DSRCT in serous effusions, with the majority of published cases depicting undifferentiated small blue cells that need to be distinguished from other small blue cell tumors. We report an interesting case of DSRCT involving a pleural effusion with a “floating island” pattern that has been described in hepatocellular carcinoma, renal cell carcinoma, and adrenal cortical carcinoma. In our case, the epithelioid tumor cells form cohesive aggregates surrounded by a single layer of spindle cells, mimicking the “endothelial wrapping” in other tumors with “floating island” patterns. We demonstrate, by ancillary testing, that these peripheral spindle cells are tapered/flattened DSRCT cells, in contrast to endothelial wrapping cells, as seen in other tumors with this unique cytomorphology. To our knowledge, this is the first case report describing DSRCT showing a “floating island” pattern that needs to be differentiated from metastatic hepatocellular carcinoma, renal cell carcinoma, and adrenal cortical carcinoma in effusion cytology.

Elevated expression of VEGFR-2 and VEGFA in desmoplastic small round cell tumor (DSRCT) and activity of bevacizumab and irinotecan in a xenograft model of DSRCT

10016 Background: Desmoplastic small round cell tumor (DSRCT) is an aggressive tumor with poor response to multimodality therapies. Given the vascular nature of many DSRCT, we sought to determine if VEGF targeting could be effective in a preclinical model of DSRCT. Methods: RNA was extracted from frozen tumor samples (DSRCT, alveolar soft part sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma, and Ewing sarcoma) and a human DSRCT cell line, JN-DSRCT. Microarray profiling utilized Affymetrix U133A/B or Affymetrix U133 Plus 2.0 chips. DSRCT xenografts were established by intramuscular injection of 20 million JN-DSRCT cells into the lower extremity of SCID/bg mice. A primary tumor developed within 8 - 12 weeks with concomitant development of abdominal metastases. Mice were treated during primary tumor growth with bevacizumab (5ug/kg IP weekly), irinotecan (2.5mg/kg IP ×10 days q3 weeks), or a combination of both. Results: Microarray data demonstrated an average of 4.5 times higher RNA expression of VEGFR-2 (KDR) in DSRCT tumor samples as compared to the other translocation-associated sarcomas (p = 3.6 E-12). VEGFR-2 was highly expressed in the JN-DSRCT line as compared to other sarcoma lines. VEGFA was also highly overexpressed in the DSRCT line and tumor samples when compared to the other translocation-associated sarcomas (2.5 times, p = 1.1E-10). Xenografts treated with bevacizumab had slowed growth over 100 days compared to control groups (volume of 0.52 mm3 vs 1.52, p = 0.002). Marked long term regressions were evident following treatment with the combination of irinotecan plus bevacizumab compared with irinotecan alone (0.12mm3 vs 0.44 at 100 days, p < 0.0001). Conclusions: VEGFR-2 and VEGFA are overexpressed in DSRCT. DSRCT xenografts were highly responsive to bevacizumab or bevacizumab plus irinotecan. Taken together, the expression data and the sustained response of DSRCT xenografts to bevacizumab suggest that VEGF-dependent angiogenesis is important for DSRCT tumor biology and represents an attractive target for therapy. These studies suggest that irinotecan and bevacizumab should be considered for inclusion in clinical trials for the treatment of DSRCT. No significant financial relationships to disclose.

Adenosine Transporter ENT4 Is a Direct Target of EWS/WT1 Translocation Product and Is Highly Expressed in Desmoplastic Small Round Cell Tumor

BackgroundDesmoplastic Small Round Cell Tumor (DSRCT) is a highly aggressive malignancy that affects mainly adolescents and young adults. A defining characteristic of DSRCT is a specific chromosomal translocation, t(11;22)(p13;q12), that fuses EWS with WT1, leading to a production of two isoforms of chimeric transcription factor, EWS/WT1(−KTS) and EWS/WT1(+KTS). The chimeric proteins are thought to play critical roles in various stages of oncogenesis through aberrant transcription of different genes, but only a few of these genes have been identified.Methodology/Principal FindingsWe report the identification of a new target of EWS/WT1, ENT4 (equilibrative nucleoside transporter 4) which encodes a pH-dependent adenosine transporter. ENT4 is transcriptionally activated by both isoforms of EWS/WT1 as evidenced by promoter-reporter and chromatin immunoprecipitation (ChIP) analyses. Furthermore, ENT4 is highly and specifically expressed in primary tumors of DSRCT as well as in a DSRCT cell line, JN-DSRCT-1. Treatment of JN-DSRCT-1 cells with adenosine analogs, such as 2-chloro-2′-deoxyadenosine (2-CdA), resulted in an increased cytotoxic response in dose- and pH-dependent manner.Conclusions/SignificanceOur detailed analyses of a novel target of EWS/WT1 in DSRCT reveal an insight into the oncogenic mechanism of EWS-fusion chromosomal translocation gene products and provide a new marker for DSRCT. Furthermore, identification of ENT4 as a highly expressed transcript in DSRCT may represent an attractive pathway for targeting chemotherapeutic drugs into DSRCT.

Androgen and c-Kit receptors in desmoplastic small round cell tumors resistant to chemotherapy: novel targets for therapy

Desmoplastic small round cell tumor (DSRCT) is a highly fatal, mainly peritoneal cell origin cancer which predominantly affects young adult males. This predilection in young males led us to examine the role of androgen receptors (AR), testosterone, and growth factors in the biology of DSRCT. Slides were prepared from 27 multi-institutional patients all with end-stage DSRCT. Slides were stained for AR, c-Kit, various growth factors, and drug resistance-associated proteins. Immunohistochemical (IHC) expression was scored semi-quantitatively. Western blot and MTT studies were performed to validate the IHC findings of over-expression of the AR and its functional status by stimulation of growth by dihydrotestosterone, respectively. Six patients with positive AR status were treated solely with combined androgen blockade (CAB) as used for prostate cancer. Twenty-two patients were male (81%) and five were female (19%) with a median age at diagnosis of 23. All patients had failed at least two prior multi-agent chemotherapy regimens and 44% had progressed after autologous stem cell transplant. DSRCT samples from 10 of 27 patients were >or=2+ IHC positive for AR (37%,P=0.0045) and 7 of 20 patients were >or=2+ IHC positive for c-Kit (35%, P=0.018). We found elevated IHC expression of GST-pi, MRP and thymidylate synthase in smaller subsets of patients. In vitro studies for AR by Western blot and stimulation of growth by dihydrotestosterone in MTT assays suggest that the AR in DSRCT cells is functional. Six patients with positive AR status were treated with CAB alone and three of six attained clinical benefit (1-PR, 1-MR, 1-SD) in a range of 3-4 months. The three patients who responded to CAB had normal testosterone levels before CAB, while the three who did not respond to CAB had baseline castrate levels of testosterone. DSRCT has significant IHC expression of AR and c-Kit in heavily pre-treated patients. The presence of significant AR expression in 37% suggests that these patients could possibly respond to CAB. The significance of c-Kit expression in 35% of DSRCT patients is unknown and warrants further investigation.