Withanolide analogues disrupt a leukemic dependency on cholesterol transport by inhibiting the oxysterol-binding protein OSBP (TPP)

Metabolic alterations in cancers precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product inspired small molecules can provide a resource of underexplored chemotypes. We identify W7, a synthetic withanolide analog with pronounced anti-leukemic properties via orthogonal chemical screening. Through multi-omics profiling (including expression proteomics) and genome-scale CRISPR/Cas9 screens, we identify that W7 disrupts Golgi homeostasis via a mechanism that requires active PI4P signaling at the ER-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of W7. Collectively, our data reaffirms sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound W7.