<i>N</i> ′-(1-Phenylethylidene)benzohydrazide Cytotoxicity Is Lysine-Specific Demethylase 1 Independent and Linked to Iron–Sulfur Cluster Disruption in Ewing Sarcoma

The noncompetitive lysine-specific demethylase 1 (LSD1) inhibitors SP-2509 and SP-2577 are <i>N</i>'-(1-phenylethylidene)-benzohydrazides that display potent activity in Ewing sarcoma. They block transcriptional regulation of the causative oncogenic fusion protein EWSR1::FLI1 and cause cell death. However, SP-2509 and SP-2577 are the only LSD1 inhibitors active in Ewing sarcoma; other LSD1 inhibitors have little effect. Studies from our group and others suggest that SP-2509 activity may result from off-target activity affecting the mitochondria. Here, we identified potential off-target mechanisms of <i>N</i>'-(1-phenylethylidene)-benzohydrazides using an unbiased approach, cellular thermal shift assay coupled to mass spectrometry. Interestingly, this revealed significant destabilization of the electron transport chain complex III protein ubiquinol-cytochrome c reductase (UQCRFS1). We find that UQCRFS1 destabilization is likely linked to impaired iron-sulfur (Fe-S) cofactor binding and that SP-2509 broadly destabilizes cellular Fe-S proteins. Using both chemical and genetic tools, we show that SP-2509 mediated cell death is LSD1 independent and instead requires a <i>N</i>'-(2-hydroxybenzylidene)-benzohydrazide. Our studies suggest that this core moiety alters iron metabolism in the cell. Importantly, we also find that the reversal of EWSR1::FLI1 transcriptional regulation by SP-2509 is independent of LSD1 inhibition. This unique activity is instead associated with the <i>N</i>'-(2-hydroxybenzylidene)-benzohydrazide core and destabilization of Fe-S proteins. These findings reveal a novel mechanism of action for this class of compounds and raise additional questions regarding how EWSR1::FLI1 transcriptional regulation is linked to Fe-S biogenesis, the precise mechanisms of cell death, the biological features of susceptible cancer cells, and strategies for clinical translation.