Abstract
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 etal., 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 invitro, 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
The case of rigosertib (ON01910) is a classic example of pleiotropic effects confounding targeted assays; depending on the type of assay, supposed evidence has emerged for multiple conflicting molecular targets
Our results strongly suggested that rigosertib kills cancer cells by directly destabilizing microtubules
Regardless, we show that expression of the L240F TUBB mutant, which we had selected due to the proximity of the L240 residue to rigosertib in our original crystal structure, conferred resistance to rigosertibpharm, strongly supporting the conclusion that rigosertib binds to tubulin in the mode we described in our original manuscript
Summary
The case of rigosertib (ON01910) is a classic example of pleiotropic effects confounding targeted assays; depending on the type of assay, supposed evidence has emerged for multiple conflicting molecular targets It is worth outlining the history of rigosertib’s development here to illustrate this issue. Rigosertib was first described by Reddy and co-workers in 2005 as an in vitro inhibitor of polo-like kinase 1 (PLK1) and proposed to kill cancer cells through this activity, based on measurements of cell cycle progression and cellular PLK1 activity (Gumireddy et al, 2005) This claim was disputed by Steegmaier et al in 2007, who found that the cellular phenotypes induced by rigosertib did not match those induced by the bona fide PLK1 inhibitor BI2536, with rigosertib’s suppression of cellular PLK1 activity likely being an indirect effect (Steegmaier et al, 2007).
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