Abstract 3489: EWSR1::WT1 fusion oncoprotein and the androgen receptor shape the biology of desmoplastic small round cell tumors

Abstract 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. Citation Format: Danh Truong, Emre Arslan, Veena Kochat, Jiaqian Fan, Diana Shamsutdinova, Davis Ingram, Kevin Murgas, Clement Agyemang, Roberto Cardenas-Zuniga, Asmaa G. Ahmed, Justin Magrath, Alexander Lazar, Najat Daw Bitar, Sean B. Lee, Andrea Hayes-Dixon, Kunal Rai, Joseph A. Ludwig. EWSR1::WT1 fusion oncoprotein and the androgen receptor shape the biology of desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3489.