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. 2020 Apr 28;9(4):41.
doi: 10.1038/s41389-020-0224-1.

Desmoplastic small round cell tumor is dependent on the EWS-WT1 transcription factor

Jenna M Gedminas  1   2   3 , Maggie H Chasse  1 , Mitchell McBrairty  1 , Ian Beddows  1 , Susan M Kitchen-Goosen  1 , Patrick J Grohar  4   5   6
Affiliations

Desmoplastic small round cell tumor is dependent on the EWS-WT1 transcription factor

Jenna M Gedminas et al. Oncogenesis. .

Abstract

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft-tissue malignancy with a poor overall survival and no effective therapeutic options. The tumor is believed to be dependent on the continued activity of the oncogenic EWS-WT1 transcription factor. However, the dependence of the tumor on EWS-WT1 has not been well established. In addition, there are no studies exploring the downstream transcriptional program across multiple cell lines. In this study, we have developed a novel approach to selectively silence EWS-WT1 without impacting either wild-type EWSR1 or WT1. We show a clear dependence of the tumor on EWS-WT1 in two different cell lines, BER and JN-DSCRT-1. In addition, we identify and validate important downstream target pathways commonly dysregulated in other translocation-positive sarcomas, including PRC2, mTOR, and TGFB. Surprisingly, there is striking overlap between the EWS-WT1 and EWS-FLI1 gene signatures, despite the fact that the DNA-binding domain of the fusion proteins, WT1 and FLI1, is structurally unique and classified as different types of transcription factors. This study provides important insight into the biology of this disease relative to other translocation-positive sarcomas, and the basis for the therapeutic targeting of EWS-WT1 for this disease that has limited therapeutic options.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. EWS-WT1 silencing leads to loss of DSRCT cell proliferation.
a Western blot showing selective suppression of EWS-WT1 expression, but not EWSR1 or WT1 at 2 nmol/L compared with untreated cells (med.), a non-targeting siRNA (siNeg), or higher concentrations of siRNA that reduce the expression of wild-type EWSR1, wild-type WT1, and EWS-WT1. b Western blot demonstrates suppression of EWS-WT1 over time (hours) following siRNA silencing with 2 nmol/L siRNA in JN-DSRCT-1 relative to untreated cells (Med) or a non-targeting siRNA (Neg). Custom siRNA (Dharmacon) targeted to flank the EWS-WT1 breakpoint was complexed with RNAiMax (ThermoFisher) at the indicated concentration for 30 min at room temperature. Cells were added and incubated at 37 °C. The cells were collected, washed in PBS, and lysed in 4% lithium dodecyl sulfate (LDS) buffer. The protein was quantitated by bicinchoninic acid (BCA) assay (Pierce, Thermo-Scientific) and 30 µg were resolved on a NuPage 4–12% Bis–Tris gradient gel (Invitrogen) in 1× NuPage MOPS SDS Running Buffer (Invitrogen). The protein was transferred overnight to a nitrocellulose membrane at 20 V in 1× Tris-Glycine-SDS Buffer (Bio-Rad) containing 20% methanol. The membrane was blocked in 5% milk in TBS-T, and probed with EWSR1 (11910, Cell Signaling) and H3 (2650, Cell Signaling), and WT1 (sc-7385, Santa Cruz Biotechnology) antibodies. The results shown are representative of three independent experiments. c, d Silencing of EWS-WT1 (siEWS-WT1) leads to a loss of proliferation as measured by percent confluence relative to the positive control (siDeath), a non-targeting siRNA (siNeg), or untreated cells (Media) in JN-DSRCT-1 (c) and BER (d) cells. JN-DSRCT-1 or BER cells were incubated with complexed siRNA/lipid at 37 °C in the Incucyte ZoomTM (Essen Bioscience) to measure confluence every 2 h. The results are displayed as the mean value with error bars representing standard deviation. Three individual replicates were plated in triplicate. t Tests were used to evaluate statistical significance from control, and adjustments were made to account for multiple comparisons. JN-DSRCT-1 cells were obtained from H. Li at New York University, and BER cells from the Christus Stehlin Foundation for Cancer Research and were confirmed mycoplasma negative. The presence of EWS-WT1 was confirmed by FISH (Supplementary Fig. 1). The cells were cultured at 37 °C, 5% CO2 in RPMI-1640 with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Fig. 2
Fig. 2. Loss of EWS-WT1 leads to morphologic changes and growth arrest in DSRCT cells.
a JN-DSCRT-1 cells undergo a morphologic change with EWS-WT1 silencing (siEWS-WT1) compared with untreated cells (Medium), a non-targeting siRNA (siNeg), or a positive control siRNA (siDeath). b, c Western blot demonstrates accumulation of the apoptotic marker cleaved PARP following EWS-WT1 silencing (b) that parallels cleaved caspase 3/7 (green cells) after EWS-WT1 silencing in live cells (c) occurring long after the morphologic change. Lysates were collected at 16, 24, 30, 48, 72, and 96 h of exposure. Western blot probed with EWSR1 (11910, Cell Signaling), H3 (2650, Cell Signaling), and cPARP (9546, Cell Signaling) and WT1 (sc-7385, Santa Cruz Biotechnology) antibodies. Apoptosis was quantitated by measuring activation of CellEvent caspase 3/7 green detection reagent (ThermoFisher Scientific) in 10,000 cells/well in a 96-well plate following siRNA silencing of EWS-WT1.
Fig. 3
Fig. 3. EWS-WT1 loss leads to large-scale gene expression changes in DSRCT cells.
a Hierarchical clustering of gene expression changes following silencing of EWS-WT1 in JN-DSRCT-1 cells (JNDSRCT1) or BER cells (BER) relative to a non-targeting control siRNA in the same cells (JN_control or BER_control). Data represent 1879 gene expression changes that are either increased (red) or decreased (blue) by a log FC of >2. b Volcano plots showing the magnitude of gene expression change as a function of q value (false discovery rate-adjusted P value) with silencing. Gene expression changes are both common (red) across the two cell lines and unique to the cell line (blue). More genes are repressed by EWS-WT1 than are induced. Libraries were prepared from 500 ng of total RNA using the KAPA-stranded mRNAseq Kit (v5.17). RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to Bio Scientific NEXTflex dual adapters. Quality and quantity were determined using a combination of Agilent DNA High Sensitivity chip, QuantiFluor® dsDNA System, and Kapa Illumina Library Quantification qPCR assays. Individually indexed libraries were pooled, and 75-bp, paired-end sequencing was performed on an Illumina NextSeq 500 sequencer using a 75-bp HO sequencing kit (v2). Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and demultiplexed to FastQ format with bcl2fastq v1.9.0 (Illumina Inc.). Reads were aligned to hg38 using STAR (v2.5.2b) with options –twopassMode Basic –quantMode GeneCounts. The data were filtered for a minimum of ten counts per million in at least one sample. Differential expression analysis was carried out using Deseq2 (v1.24.0) with apeglm (v1.6.0) applied to account for absolute magnitude of gene expression. Significant genes were determined using a cutoff of q value < 0.05. Heatmaps were generated using pheatmap package (v1.0.12) in R (v3.6.1). Analysis was performed on three biological replicates of each sample. All samples were collected at the same time, and RNA was isolated on the QiaCube (Qiagen) in batches after sample randomization to avoid batch effect. The defined EWS-WT1 gene signature consists of genes with a log-fold change of 2 or greater in both cell lines following silencing. c Western blot of EWS-WT1 silencing confirms repression (ERG) and induction (CEBPD) of targets. Lysates collected at 16, 24, 30, and 48 h of exposure. Western blot probed with EWSR1 (11910, Cell Signaling) and H3 (2650, Cell Signaling), WT1 (sc-7385 Santa Cruz Biotechnology), ERG (ab92513, Abcam), and CEBPD (ab65081, Abcam) antibodies.
Fig. 4
Fig. 4. Identification of therapeutic vulnerabilities using functional gene set enrichment analysis (fgsea).
a fgsea analysis of the top ten differentially regulated pathways in DSRCT cells following EWS-WT1 silencing demonstrates enrichment of cell-cycle and DNA repair pathways and highlights MYC signaling. There is also enrichment of gene sets that share similarities with the EWS-FLI1 transcription factor, including PRC2 complex-associated pathways, E2F, and TGFB. b Heatmaps showing either induction (red) or suppression (blue) of expression of known EWS-FLI1-induced (left) or repressed (right) targets with silencing of EWS-WT1 in DSRCT cells. Gene set enrichment analysis (fGSEA) was performed with functional gsea package (v1.10.1), and heatmaps were generated using pheatmap package (v1.0.12) in R (v3.6.1). c Silencing of ERG (siERG) leads to loss of cellular proliferation, which parallels that following EWS-WT1 silencing (siEWS-WT1) measured by the total cell number using MTS assay. Cell viability was measured using CellTiter96 (Promega) in three separate replicates plated in triplicate. d Silencing of ERG (siERG) leads to the accumulation of the apoptotic marker cleaved caspase similar to that seen following EWS-WT1 silencing (siEWS-WT1) measured using green fluorescent-cleaved caspase 3/7. siRNA and apoptosis measurement were completed as described above.
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