A Family of Glycosylated Macrolides Selectively Target Energetic Vulnerabilities in Leukemia

Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. CryoEM of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence define the mechanism of action of apoptolidin family glycomacrolides and establish a path to address OXPHOS-dependent cancers. ### Competing Interest Statement M.R.S. receives research funding from Astex, Incyte, Takeda, and TG Therapeutics; has equity with Karyopharm; serves as advisory or consultant to AbbVie, Astex, BMS, Celgene, Incyte, Karyopharm, Ryvu, Sierra Oncology, Takeda, TG Therapeutics. P.B.F currently receives research funding from Incyte, and has received research funding from Astex and Forma Therapeutics in the past.