Abstract 1436: The nonenzymatic effects of LSD1

Abstract Ewing sarcoma is an aggressive malignancy and the second most frequent bone tumor afflicting the adolescent and young adult population. Despite knowing the oncologic driving event of Ewing sarcoma for decades, outcomes and treatment options for this disease have not improved. A chromosomal translocation between chromosome 11 and 22, fusing the EWSR1 gene to the FLI1 gene (EWSR1::FLI1), is the cause of eighty five percent of Ewing sarcoma cases. Due to the inability to therapeutically target EWSR1::FLI1, the field has instead attempted to target critical co-regulators to disrupt the oncogenic program of EWSR1::FLI1. Histone specific lysine demethylase 1 (LSD1) is overexpressed in Ewing sarcoma and higher levels of LSD1 correlate with poor patient prognosis. Furthermore, LSD1 directly interacts with, colocalizes with, and targets a similar gene set as EWSR1::FLI1, making it a promising target for targeted therapy. Multiple LSD1 inhibitors have been studied with varying effects on the Ewing transcriptome. In particular, OG-L002, a tranylcypromine derivative with potent and specific LSD1 inhibition has little effect on Ewing sarcoma cells, while noncompetitive small molecule inhibitors targeting enzymatic and nonenzymatic functions of LSD1 kill Ewing sarcoma cells at sub micromolar concentrations. As such, we hypothesize that LSD1 has important nonenzymatic functions in Ewing sarcoma. We have curated a suite of molecular tools to deplete LSD1 DNA, mRNA, and protein to define the nonenzymatic effects of LSD1 in Ewing sarcoma. We have knocked out LSD1 via CRISPR genome editing at the DNA level, degraded LSD1 mature RNA via short hairpin RNA interference, degraded LSD1 protein with the UM171 protein degrader, and enzymatically inhibited LSD1 with OG-L002. In every condition we measured the effect on cellular proliferation with Incucyte growth assays, tested the impact on anchorage independent growth with soft agar transformation assays, and found the differentially expressed transcripts with RNA-sequencing. There are only 4 differentially expressed genes when cells are treated with the enzymatic inhibitor OG-L002, compared to hundreds of differentially expressed genes when nonenzymatic functions are depleted as well. In addition, 39 genes that are commonly targeted by depletion at the DNA, RNA, and protein level, corresponding to genes regulated by the nonenzymatic functions of LSD1. We rescued the LSD1 knock out cells with an enzymatically dead mutant (LSD1 K661A) to further specify genes regulated by LSD1 nonenzymatic functions. These genes are related to transcription factor coregulation, axon development, and synaptic organization and signaling. We believe LSD1 serves a scaffolding or recruitment role in various larger chromatin regulatory complexes that are essential for regulation of these genes in Ewing sarcoma oncogenesis that is distinct from LSD1 demethylase activity. Citation Format: Rachel D. Dreher, Ira Miller, Emily R. Theisen. The nonenzymatic effects of LSD1 [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 1436.