(P 23) DEFINING THE NONENZYMATIC FUNCTIONS OF LSD1 IN EWING SARCOMA

Objective: Ewing sarcoma (ES) 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 ES for decades, outcomes and treatment options have not improved. A translocation between chromosome 11 and 22, fusing the EWSR1 gene to the FLI1 gene (EWS::FLI1), is the cause of 85% of ES cases. Due to the inability to therapeutically target EWS::FLI1, targeting critical co-regulators of EWS::FLI1 is a more promising therapeutic avenue. One such co-regulator, histone specific lysine demethylase 1 (LSD1), is overexpressed in ES, directly interacts with, colocalizes with EWS::FLI1. Multiple LSD1 inhibitors have been developed, but the efficacy of these inhibitors has been challenging to interpret. Enzymatic inhibition seems to have no impact on cells, but noncompetitive inhibition results in significant off target effects. We hypothesize that LSD1 regulates a distinct set of genes via its nonenzymatic functions in ES.

Methods: To pinpoint the nonenzymatic effects of LSD1, we have depleted LSD1 at the DNA level via CRISPR genome editing and the protein level with UM-171 in 3 ES cell lines. We rescued LSD1 with a wildtype construct or a construct with two key residues mutated, K661Q and A539E, rendering the protein enzymatically dead. We tested for proliferation and transformation capacity defects via Incucyte growth assays and soft agar transformation assays, then found differentially expressed transcripts with RNA-sequencing.

Results: Of note, only 4 genes are differentially expressed when cells enzymatic inhibited by OG-L002. With loss of the complete protein by CRISPR or degradation, hundreds of genes are differentially expressed. These genes are involved in axon development and neural differentiation pathways, correlating with known LSD1 functions.

Conclusion: Our results suggest the demethylase activity of LSD1 is dispensable, whereas the nonenzymatic functions have critical gene targets in ES. LSD1 likely serves a scaffolding or recruitment role, distinct from its demethylase activity, in larger chromatin regulatory complexes that significantly contributes to the ES genetic program.