Transcriptome analysis of desmoplastic small round cell tumors identifies actionable therapeutic targets: a report from the Children’s Oncology Group

Abstract

to further understand the molecular pathogenesis of desmoplastic small round cell tumor (DSRct), a fatal malignancy occurring primarily in adolescent/young adult males, we used next-generation RNA sequencing to investigate the gene expression profiles intrinsic to this disease. RNA from DSRCT specimens obtained from the Children's Oncology Group was sequenced using the Illumina HiSeq 2000 system and subjected to bioinformatic analyses. Validation and functional studies included WT1 ChIP-seq, EWS-WT1 knockdown using JN-DSRCT-1 cells and immunohistochemistry. A panel of immune signature genes was also evaluated to identify possible immune therapeutic targets. Twelve of 14 tumor samples demonstrated presence of the diagnostic EWSR1-WT1 translocation and these 12 samples were used for the remainder of the analysis. RNA sequencing confirmed the lack of fulllength WT1 in all fusion positive samples as well as the JN-DSRCT-1 cell line. ChIP-seq for WT1 showed significant overlap with genes found to be highly expressed, including IGF2 and FGFR4, which were both highly expressed and targets of the EWS-WT1 fusion protein. In addition, we identified CD200 and CD276 as potentially targetable immune checkpoints whose expression is independent of the EWS-WT1 fusion gene in cultured DSCRT cells. In conclusion, we identified IGF2, FGFR4, CD200, and CD276 as potential therapeutic targets with clinical relevance for patients with DSRct. DSRCT was first described by Gerald and Rosai 1 as a highly malignant soft-tissue sarcoma occurring in adolescent and young adults, especially in males. It often presents with widespread disease with malignant foci present throughout the peritoneal cavity. Despite intense multi-agent chemotherapy, surgery, radiation therapy, and incorporation of other treatment modalities like high-dose chemotherapy with autologous stem cell rescue, 5-year event-free survival remains dismal at less than 20%, while the median survival time is approximately 2.5 years 2. Novel therapies are therefore urgently needed to improve the outcomes for patients with this disease.

Establishment of multiple novel patient-derived models of desmoplastic small round cell tumor enabling functional characterization of ERBB pathway signaling and pre-clinical evaluation of a novel targeted therapy approach

“…We next performed immunohistochemistry on paraffin-embedded DSRCT cell line xenografts or cell pellets (SK-DSRCT1) to confirm the presence of WT1 using an antibody raised to the C-terminus of WT1, and to explore expression of EGFR and phosphorylated EGFR, given that this ERBB RTK was the most consistently activated in cell lines and tumors tested. Strong nuclear staining for the WT1 C-terminus was detected in all samples probed, most likely representing EWSR1-WT1 in these novel DSRCT models, as a recent transcriptome profiling study of has confirmed a lack of native WT1 transcripts in DSRCT (35). EGFR was detected at varying levels in the different DSRCT patient xenografts or xenografted DSRCT cell lines.…”

“…Western blotting with the C-terminal WT1 antisera confirmed the presence of WT1 in all of the new DSRCT cell lines ( Figure 3D ). The band detected is most likely EWS-WT1 as transcriptomic analysis has shown that the JN-DSRCT-1 cell line does not express wildtype WT1 (35).…”

Desmoplastic Small Round Cell Tumor: A Review of Main Molecular Abnormalities and Emerging Therapy

“…Silencing EWS-WT1 causes proliferation loss, growth arrest and gene expression analysis indicates a repression of estrogen signaling and highlight therapeutic genetic vulnerabilities, such as FGFR4, JAK3, mTOR, PDGF, ERG, and TGFB1 genes [12]. Another study that evaluated potential therapeutic targets performed RNA sequencing of 12 tumor samples from pediatric patients with DSRCT found high expression of IGF2, FGFR4, CD200 and CD276, the latter two molecules are candidates for immune checkpoint inhibitor therapy [75] In terms of therapy, the first use of JN-DSRCT-1 cells was to test de effect of rapamycin induced in inducing apoptotic death [76]. The mechanism involves the up-regulation of Bax concomitant Bcl-xL down-regulation.…”

“…gene promoter, providing the basis to test the activity of anti-IGF-1R antibodies in the metastatic setting [119]. Also, IGF2 has been up-regulated by the fusion product and is a potential target [75].…”

“…However, preclinical data using gene expression analysis showed that DSRCT overexpress immune regulatory proteins such as CD200 and CD276 (B7H3), which is not regulated by the EWS-WT1 fusion protein [75]. Currently, enoblituzumab is being tested in many solid tumors (NCT02982941),…”