Abstract 6932: Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in desmoplastic small round cell tumors

Abstract Background: Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, aggressive sarcoma primarily affecting young males. Despite being driven by the unique oncogenic transcription factor (TF) EWSR1-WT1, and a monotonous morphology, DSRCT cells display a polyphenotypic differentiation of unknown etiology. Methods: To explore DSRCT heterogeneity, we characterized a series of patient samples by single-cell, spatial, and bulk multi-omics. We integrated patients’ data with bulk RNA-sequencing, ChIP-sequencing, and ATAC-sequencing data generated in a DSRCT cell line model upon EWSR1-WT1 transcriptional silencing. Results: Single-cell RNA-sequencing (scRNA-seq) on 11 patient samples identified recurrent subpopulations of DSRCT tumor cells, including lineage-, metabolism-, and pseudostate-related clusters. These subpopulations showed overlapping gene modules and lacked copy number variation diversity, suggesting transcriptional plasticity as a potential source of tumor heterogeneity. We first investigated whether cell-intrinsic mechanisms may drive DSRCT intra-tumor heterogeneity. Using an in-house-developed assay, we found limited variability in EWSR1-WT1 transcript expression. Preclinical modeling defined an EWSR1-WT1 regulon, used as a surrogate for EWSR1-WT1 TF activity. Integration with scRNA-seq data showed that high EWSR1-WT1 activity was associated with lineage-related states, while glycolytic and profibrotic states correlated with low EWSR1-WT1 activity. Single-cell ATAC-seq with scRNA-seq (Multiome) on one patient sample identified TF motifs related to developmental processes - e.g., SOX, FOX, and GATA - as the most differentially enriched across clusters. Interestingly, EGR1 regulon activity showed an inverse correlation with EWSR1-WT1 and Androgen Receptor regulons’ activity, in line with preclinical data, where ATAC-seq showed increased accessibility of EGR1 upon EWSR1-WT1 silencing. We next investigated cell-extrinsic factors by spatial transcriptomics in six patient samples. Glycolytic and profibrotic subpopulations localized within hypoxic niches at the tumor periphery, in direct proximity to cancer-associated fibroblasts (CAFs), which promoted tumor growth in vitro, as assessed in DSRCT cells/CAFs co-culture assays. Finally focusing on inter-tumor heterogeneity, the prognostic evaluation of scRNA-seq-derived signatures revealed that a tumor cell epithelial signature correlated with improved patient survival in a cohort of 29 DSRCT samples profiled with bulk RNA-sequencing. Conclusion: DSRCT heterogeneity is driven by combined cell-intrinsic and -extrinsic mechanisms, including EWSR1-WT1 DNA-binding activity and interaction with CAFs. Gene signatures associated with specific DSRCT subpopulations correlated with prognosis, highlighting the clinical relevance of our findings. Citation Format: Clémence Henon, Julien Vibert, Thomas Eychenne, Nadège Gruel, Léo Colmet-Daage, Carine Ngo, Marlène Garrido, Nicolas Dorvault, Maria Eugenia Marques Da Costa, Antonin Marchais, Asuka Kawachi, Madison Lenormand, Clémence Astier, Roman Chabanon, Benjamin Verret, Rastislav Bahleda, Axel Le Cesne, Fatima Mechta-Grigoriou, Charles Honoré, Matthieu Faron, Olivier Delattre, Joshua J Waterfall, Sarah Watson, Sophie Postel-Vinay. Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6932.