LSD1 Performs Demethylase-Independent and Context-Specific Roles in Ewing Sarcoma

Lysine specific demethylase 1 (LSD1), encoded by the gene <i>KDM1A</i>, is overexpressed and correlates with poor patient prognosis in Ewing sarcoma. LSD1 and the pathognomonic fusion oncoprotein, EWSR1::FLI1, colocalize throughout the genome, suggesting LSD1 is a critical co-regulator driving the progression of Ewing sarcoma. However, therapeutic targeting of LSD1 by competitive and noncompetitive inhibitors has yielded mixed results. Irreversible, enzymatic inhibition seems ineffective, but reversible noncompetitive inhibition has predominant off target mechanisms, leaving open the question of LSD1 function in Ewing sarcoma. Here we take a robust approach through multiple methods of depletion in multiple EwS cell lines to define enzymatic and nonenzymatic contributions of LSD1 to transcriptional regulation. We define a core set of 22 genes that are commonly repressed by LSD1 in all cell lines, and that repression of these genes downregulates synapse functioning and e-cadherin target genes. Derepression of these genes with LSD1 loss is an early and sustained genotype in all cell lines tested. We further define distinct gene sets in each cell line that are regulated by enzymatic and nonenzymatic LSD1 activity and find repression of e-cadherin target genes to be nonenzymatically regulated. This finding supports the growing body of evidence that in addition to their canonical catalytic activity, chromatin regulatory enzymes serve essential noncanonical roles as well. Furthermore, we uncovered evidence through use of the irreversible inhibitor OG-L002 that 2D cytotoxicity and proliferation assays may be insufficient to determine Ewing sarcoma response to LSD1 inhibition.