EWSR1::WT1 fusion oncoprotein and the androgen receptor shape the biology of desmoplastic small round cell tumors

Desmoplastic small round cell tumors (DSRCTs) are rare, aggressive sarcomas driven by the EWSR1::WT1 fusion oncoprotein (FP) and marked by poor long-term survival. Despite a relatively quiet genome with a single known oncogenic driver, DSRCTs display striking transcriptional heterogeneity and express markers from multiple lineages, epithelial, mesenchymal, myogenic, and neural. DSRCTs show a strong male predominance, suggesting a potential role for sex-specific regulatory mechanisms, including androgen receptor (AR) signaling. To investigate the molecular basis of this complexity, we generated a comprehensive, multi-modal atlas of DSRCT, integrating transcriptomic, epigenomic, and spatial omics data (10x Genomics). We focused on dissecting the regulatory architecture shaped by the EWSR1::WT1 fusion and transcription factors such as AR, aiming to uncover mechanisms of lineage specification and therapeutic vulnerabilities. We hypothesized that epigenetic regulation of the EWSR1::WT1 FP contributes to this lineage divergence. To explore FP activity, we found that neogenes (non-coding RNA FP targets) and known FP-regulated genes were more highly expressed in NE-lineage tumors, suggesting that FP affects lineage type. Spatial transcriptomic analysis revealed intratumoral heterogeneity, with distinct clusters of epithelial (AR+, cytokeratin+) and non-epithelial tumor cells. Despite phenotypic differences, all tumor cells expressed neogenes, as confirmed by RNA in situ hybridization (RNAScope). Epigenetic profiling showed enrichment of Forkhead TFs (FOXA1/2). FOXA2 was associated with NE, and AR/GRHL2 were associated with epithelial lineages. CUT&RUN analysis of AR after DHT stimulation revealed co-occupancy of FOXA1/2 and WT1 at AR-bound regions, suggesting interaction between AR signaling, FP, and FOXA1/2. FP knockdown in DSRCT cell lines induced a shift toward an epithelial-like state, increased FOXA1, and decreased FOXA2 expression. Tumor cells exhibit both inter- and intra-tumoral heterogeneity, adopting epithelial and NE phenotypes. We find that the high FP levels favor a NE-lineage state mediated by chromatin accessibility and transcriptional regulation. Spatial analyses revealed co-existing epithelial and non-epithelial tumor cells within the same nests, underscoring the intratumoral heterogeneity. The interaction between FP, AR, and Forkhead transcription factors (FOXA1/2) suggests a potential mechanism by which oncogenic FPs and lineage-defining transcription factors converge to regulate tumor phenotype.