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bioRxiv · 10.1101/2025.06.20.660795

N'-(1-phenylethylidene)-benzohydrazide cytotoxicity is LSD1 independent and linked to Fe-S cluster disruption in Ewing sarcoma

Sherman, J. W.·Rask, G. C.·Xiong, B.·Tokarsky, E. J.·Duncan, A.·Weerapana, E.·Theisen, E. R.
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Abstract

The noncompetitive LSD1 inhibitors SP-2509 and SP-2577 are N-(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 SP-2509 activity may result from off-target activity affecting the mitochondria. Here we identified potential off-target mechanisms of N-(1-phenylethylidene)-benzohydrazides using an unbiased approach, cellular thermal shift assay coupled to mass spectrometry (CETSA-MS). 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 N-(2-hydroxybenzylidene)hydrazide. Our studies suggest 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 from LSD1 inhibition. This unique activity is instead associated with the N-(2-hydroxybenzylidene)-hydrazide 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.

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Sherman, J. W., Rask, G. C., Xiong, B., Tokarsky, E. J., Duncan, A., Weerapana, E., Theisen, E. R.. 2025-06-26. N'-(1-phenylethylidene)-benzohydrazide cytotoxicity is LSD1 independent and linked to Fe-S cluster disruption in Ewing sarcoma. https://doi.org/10.1101/2025.06.20.660795

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