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Pharmaceutical-grade rigosertib destabilizes microtubules in cells and in vitro, and expression of a tubulin variant with a mutation in the rigosertib-binding pocket allows cells to proliferate in the presence of rigosertib. Thus, pharmaceutical-grade rigosertib kills cancer cells by directly destabilizing microtubules, contrary to the claims by Reddy et al., Summary We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings., Highlights • Pharmaceutical-grade rigosertib kills cells by destabilizing microtubules • Pharmaceutical-grade rigosertib destabilizes microtubules in cells and in vitro • A rigosertib-binding mutation in tubulin alleviates rigosertib-mediated toxicity

Summary Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib’s target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents., Graphical Abstract, Highlights • Combined CRISPRi/a chemical-genetic screening reveals targets of therapeutic agents • Focused chemical-genetic profiling rapidly classifies agents by mechanism of action • Chemical-genetic screens with rigosertib reveal a microtubule-destabilizing signature • Targeted in vivo and in vitro approaches confirm rigosertib’s mechanism of action, Jost et al. present a two-tiered strategy to identify molecular targets of bioactive compounds using CRISPRi/a-mediated chemical-genetic screens. Application to rigosertib, an anti-cancer drug with an unclear mechanism of action, points to rigosertib being a microtubule-destabilizing agent. Targeted cell biological, biochemical, and structural approaches confirm this mechanism of action.