(P 47) EWSR1::WT1 FUSION ONCOPROTEIN ACTIVITY SHAPES LINEAGE HETEROGENEITY IN DESMOPLASTIC SMALL ROUND CELL TUMORS

Objective: Desmoplastic small round cell tumors (DSRCTs) are rare, aggressive sarcomas driven by the EWSR1::WT1 fusion oncoprotein (FP) and characterized by poor long-term survival. To uncover mechanisms underlying tumor plasticity and therapeutic resistance, we investigated how the FP may drive lineage through integrated multi-omic analyses.

Methods: We performed scMultiome on post-chemotherapy DSRCT specimens to assess gene expression and chromatin accessibility. Androgen receptor (AR) CUT&RUN after dihydrotestosterone (DHT) stimulation and HOMER motif analysis explored transcriptional regulation. Spatial heterogeneity and FP activity were evaluated using multiplex immunofluorescence and RNAScope.

Results: ScMultiome analysis revealed inter-tumoral heterogeneity, distinguishing tumors by epithelial and neural (NE) lineage signatures, with epithelial lineage co-expressing AR. 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 FP effects on 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 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.

Conclusion: 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, highlighting the pervasive role of the FP. Differences in cell states may influence chemosensitivity, and targeting cell state transitions may present a novel therapeutic approach. Our work also suggests that future FP-directed therapy will alter cell fate and phenotype.