Abstract 5947: LSD1 performs demethylase-independent and context-specific functions in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is an aggressive malignancy afflicting adolescents and young adults. A chromosomal translocation fusing the EWSR1 gene to the FLI1 gene (EWSR1::FLI1) causes 85% of cases. Due to the inability to target EWSR1::FLI1, the field has attempted to target critical co-regulators to disrupt oncogenesis. One candidate is Lysine specific demethylase 1 (LSD1), which is overexpressed in EwS, colocalizes with the fusion, and inversely correlates with patient prognosis. LSD1 inhibitors have shown mixed activity in EwS. Noncompetitive LSD1 inhibitors have potent cytotoxic activity, but these effects are not seen with irreversible inhibitors. Recent work from our group suggests the activity of noncompetitive inhibitors is likely LSD1-indpendent. This has left the precise function of LSD1 in EwS unclear. We hypothesize that LSD1 has important nonenzymatic functions in EwS. We have developed a suite of genetic and pharmacological tools to deplete LSD1 and define its nonenzymatic functions. Genetically, we knocked out LSD1 via CRISPR genome editing. Pharmacologically, we degraded full-length LSD1 protein with UM171. We assessed proliferation and transformation capacity and identified differentially expressed genes with RNA sequencing. To distinguish between enzymatic and nonenzymatic functions, we also used genetic and pharmacological approaches. We engineered a full length, wild type LSD1 (LSD1wt) and a K661Q/A539E mutant enzymatically dead LSD1 (LSD1ed). Pharmacologically, we treated parental cells with irreversible inhibitor OG-L002. RNA-sequencing was used to define genes regulated by enzymatic and nonenzymatic functions. Surprisingly, LSD1 KO does not impair proliferation or anchorage independent growth. In contrast, degradation of LSD1 protein caused a pronounced, cell line-dependent reduction in colony formation. This cell line-specific reduction in colony formation was recapitulated with OG-L002 treatment. Overlap and pathway analysis found that LSD1 has both shared and cell line-specific targets. Across all cell lines, LSD1 consistently represses genes involved in neurotransmitter functioning and e-cadherin targets. In A673 cells, LSD1wt and LSD1ed demonstrated highly overlapping expression profiles. In addition, OG-L002 treatment showed no phenotype, suggesting that LSD1’s enzymatic activity is dispensable for core functions in A673. When we define nonenzymatic functions pharmacologically, we see that LSD1 performs core functions through both enzymatic and nonenzymatic means. Interestingly, repression of e-cadherin target genes is consistently a nonenzymatic function. Our results demonstrate that LSD1 has a core set of functions that it regulates via cell line dependent mechanisms. In addition, the changes seen in 3D assays with LSD1 inhibition and depletion suggest LSD1 plays a more important role in 3D growth and that future studies should prioritize 3D assays. Citation Format: Rachel D. Dreher, Cenny C. Taslim, Ira Miller, John W. Sherman, Ariunaa Bayanjargal, Emily R. Theisen. LSD1 performs demethylase-independent and context-specific functions in Ewing sarcoma 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 5947.