Abstract 4045: Leiomyosarcoma is characterized by two distinct subtypes driven by NFI or AP1 transcription factors

Abstract Objective: Leiomyosarcoma (LMS) is a smooth muscle-derived tumor documented to display significant heterogeneity. Although bulk transcriptome studies have been reported in LMS, single-cell characterization has been lacking to assess intra-tumoral cellular heterogeneity. We aimed to characterize heterogeneity, create a single-cell atlas, define molecular subtypes, and identify gene regulatory networks underlying the heterogeneity of LMS. Methods: We performed single nucleus multiome sequencing with concurrent RNA sequencing (snRNA-seq) and assay for transposase-accessible chromatin (snATAC-seq) using 16 fresh frozen, untreated LMS samples (9 retroperitoneal/vascular, 4 uterine, and 3 extremity). We delineated malignant cells from benign cells using InferCNV and CytoTRACE analyses. Spatial transcriptomic analysis was performed on 12 LMS tissue microarrays (∼250 total tissue cores, mostly pre-treated tumors) using 10x Xenium and a custom 480-gene panel informed by snRNA-seq data. Human LMS cell lines were employed to functionally validate findings from these data. Results: We obtained high-quality data from 94,439 nuclei for snRNA-seq and 89,702 nuclei for snATAC-seq. Malignant cells were characterized by a less differentiated state by CytoTRACE and higher CNVs. UMAP analysis identified 15 different cell clusters including 12 malignant cell clusters and 3 non-malignant cell clusters. Some malignant cell clusters showed signs of aberrant differentiation with enrichment of osteoblastic and neural pathways. Malignant cells fell almost exclusively into 2 distinct subtypes: a dedifferentiated subtype with mesenchymal features (MES) and a differentiated subtype with enrichment of smooth muscle cell markers (SMC). Integration of chromatin accessibility data showed enrichment of nuclear factor I (NFI) transcription factor (TF) motifs in the MES and AP-1 (BACH2, FOSB, and FOSL2) motifs in the SMC group. Whole genome sequencing did not reveal an obvious genomic etiology for these subtypes. Expression of these TFs was observed in LMS cell lines. Depletion of NFI TFs, namely NFIX, reduced proliferation and invasion of LMS cell lines. Spatial transcriptomics identified spatial relationships between these subtypes and infiltrating immune cells. This revealed a greater tendency for immunosuppressive macrophages to cluster around MES cells compared to SMC cells. Conclusion: We produced an atlas of the LMS single-cell transcriptome and epigenome. We demonstrate the presence of two predominant malignant cell clusters with dedifferentiated/mesenchymal or smooth muscle cell-like properties, a novel finding in LMS, and implicate NFI and AP-1 TFs in driving this heterogeneity. Citation Format: Ryan A. Denu, Zhao Zheng, Yingda Jiang, Veena Kochat, Emre Arslan, Danh D. Truong, William Padron, Davis R. Ingram, Khalida M. Wani, Diana Shamsutdinova, Ravin Ratan, Neeta Somaiah, Christina L. Roland, Pamela T. Soliman, Larissa A. Meyer, Alexander J. Lazar, Elise F. Nassif Haddad, Emily Z. Keung, Kunal Rai. Leiomyosarcoma is characterized by two distinct subtypes driven by NFI or AP1 transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4045.