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Abstract Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive sarcoma subtype that is usually uncurable. All malignant cells harbor a pathognomonic EWSR1: : WT1 fusion protein (FP), but FP-targeted agents are nonexistent, and less than 20% of patients survive beyond five years. Lacking the availability of FP-targeted drugs, we seek to understand how the FP regulates tumor cells to promote the characteristic multilineage phenotypes of epithelial and neuroendocrine (NE) cell fates to discover a therapeutic opportunity. Herein, we share the results of gene expression and chromatin accessibility analyses to explore fundamental mechanisms explaining epithelial and NE biology in DSRCT. We profiled 85, 640 nuclei from fresh-frozen tissue of nine DSRCT patients post-chemotherapy using single-nucleus RNA-sequencing (snRNA-seq) and ATAC-sequencing (snATAC-seq). A subset of DSRCT patients with low or negative expression of AR expressed partial NE reprogramming, akin to NE prostate cancer (NEPC), exhibiting NE markers (SYP, ENO2, CHG, FOXA2, ASCL1, and SOX2). Another patient subset expressed epithelial markers (MUC1, MUC6, KRT18, KRT23, CDH1). One specimen appeared to exhibit both markers - implying a hybrid or a poorly differentiated phenotype. This suggests that DSRCT could be classified into three subtypes: 1) Epithelial/AR-positive, 2) NE-positive, and 3) hybrid AR/NE. We used CytoTRACE to predict differentiation states. As expected, normal cells (i. e. , fibroblasts and immune cells) were the most differentiated. Neoplastic cells, identified by EWSR1: : WT1 gene targets and copy number variations, had lower differentiation scores, indicative of a less differentiated phenotype. The NE subtype had the lowest differentiation score indicative of open chromatin and greater plastic capacity, previously implicated in NEPC. SnATAC-seq revealed that the WT1 motif was enriched in malignant cells. Epithelial/AR-positive subtypes were enriched in DNA-response elements for nuclear receptors (ARE, GRE, and PGR) and the forkhead box family of transcription factors (FOXA1, a known pioneer factor for AR). The NE subtype lacked the ARE, GRE, and PGR motifs but was enriched with FOXA1 and neural-specific motifs, including NEUROD1, ATOH1, OLIG2, and NANOG. We show significant heterogeneity in DSRCT phenotypes, where we observe both epithelial and NE lineages. Initially, we detected the Epithelial/AR-positive and NE-positive lineages by gene expression, but the snATAC-seq data also supports this. Although the emergence of the NE subtype in prostate cancer is generally in response to therapy, how it emerges in DSRCT is yet unknown. The CytoTRACE analysis suggests that NE dedifferentiated from the more differentiated Epithelial/AR-positive subtype, but this remains an active area of investigation. Finally, we are working to understand the Epithelial/AR and NE in DSRCT dimorphic states. Citation Format: Danh Truong, Emre Arslan, Veena Kochat, Sandhya Krishnan, Clement Agyemang, Margarita Divenko, Davis Ingram, Rossana Lascano, Akshay Basi, Javier Gomez, Hannah Beird, Chia-Chin Wu, Jared Burks, P Andrew Futreal, Alexander Lazar, Ravin Ratan, Najat Daw, Andrea Hayes, Kunal Rai, Joseph Ludwig. Multi-omic analysis of desmoplastic small sound cell tumors reveal distinct epithelial and neuroendocrine lineages 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 (6Suppl): Abstract nr 130.
Truong et al. (Fri,) studied this question.