{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:13:32Z","timestamp":1785402812250,"version":"3.56.0"},"posted":{"date-parts":[[2025,6,26]]},"group-title":"Pharmacology and Toxicology","reference-count":44,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T00:00:00Z","timestamp":1750896000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["T32CA269052"],"award-info":[{"award-number":["T32CA269052"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["RSG-22-118-01-DMC"],"award-info":[{"award-number":["RSG-22-118-01-DMC"]}],"id":[{"id":"https:\/\/ror.org\/02e463172","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM134964"],"award-info":[{"award-number":["R35GM134964"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/01f6q3121","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/04jme7072","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,6,26]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>The noncompetitive LSD1 inhibitors SP-2509 and SP-2577 are N\u2019-(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\u2019-(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\u2019-(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\u2019-(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.<\/jats:p>","DOI":"10.1101\/2025.06.20.660795","type":"posted-content","created":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T16:00:16Z","timestamp":1750953616000},"source":"Crossref","is-referenced-by-count":0,"title":["N\u2019-(1-phenylethylidene)-benzohydrazide cytotoxicity is LSD1 independent and linked to Fe-S cluster disruption in Ewing sarcoma"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7998-9979","authenticated-orcid":false,"given":"John W.","family":"Sherman","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6877-4796","authenticated-orcid":false,"given":"Galen C.","family":"Rask","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-9602-5489","authenticated-orcid":false,"given":"Bingcong","family":"Xiong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4861-2412","authenticated-orcid":false,"given":"E. 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