Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent

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 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.

Keywords: CRISPRa; CRISPRi; chemical genetics; drug mechanism of action; drug target identification; microtubules; rigosertib.

Figure 1

Internally Controlled Sensitivity Assays to Determine Effects of KIF2C or TACC3 Knockdown or Overexpression on Sensitivity to Rigosertib_pharm, Rigosertib_comm, or ON01500 (A) CRISPRi drug sensitivity phenotypes for indicated sgRNAs. (B) CRISPRa drug sensitivity phenotypes for indicated sgRNAs. Enrichment is defined as ratio of sgRNA-positive cells to sgRNA-negative cells, normalized to the corresponding ratio after treatment with DMSO. n.d.: phenotype not determined because total counted cell numbers were < 2,500. Data represent mean and individual measurements of replicate treatments (n = 2).

Figure 2

Rigosertib Inhibits Microtubule Growth in Cells Microtubule growth speeds measured in untreated cells or cells treated with 2 μM rigosertib_pharm for 1 h. Untreated, n = 21; rigosertib, n = 29. Boxes denote IQR, central lines denote median values, whiskers denote lowest/highest datum within lower/higher quartile ± 1.5 IQR. Indicated p value derived from a one-sided Mann-Whitney U test.

Figure 3

Rigosertib_pharm Destabilizes Microtubules In Vitro (A and B) Quantification of (A) microtubule growth rate and (B) catastrophe frequency with 15 μM tubulin along with EB3 (20 nM) without or with 10 or 20 μM rigosertib_pharm. n = 40 for each condition. Boxes denote IQR, central lines denote median values, whiskers denote lowest/highest datum within lower/higher quartile ± 1.5 IQR. Indicated p values derived from one-sided Mann-Whitney U tests.

Figure 4

Expression of L240F TUBB Confers Resistance to Rigosertib_pharm (A) Log₂ enrichment of K562 cells expressing L240F TUBB or an empty construct after treatment with rigosertib_pharm or ON01500 in internally controlled growth assays. Enrichment was measured as the ratio of mCherry-positive to mCherry-negative cells, e = fraction(mCh⁺) / fraction(mCh⁻), by flow cytometry, calculated relative to the first time point. Relative enrichment for each time point was normalized to that of DMSO-treated control cells. Data represent mean and individual measurements of replicate treatments (n = 2). (B) Cumulative cell doublings of L240F TUBB-transduced or non-transduced K562 subpopulations treated with rigosertib_pharm or ON01500. Cumulative doublings were calculated from measurements of cell numbers and the fractions of mCherry-positive (L240F TUBB-transduced) and mCherry-negative cells (non-transduced) in the population. Data represent mean and individual measurements of replicate treatments (n = 2). Traces for DMSO-treated cells are identical in both panels in (B).

Figure 5

Reanalysis of the Crystal Structure of the Tubulin-Rigosertib Complex (A) Electron density of region in question after refinement of rigosertib against deposited data. 2F_o–F_c (blue) and F_o–F_c (green/red) are contoured at the indicated levels. (B) Electron density of region in question after refinement of ON01500 against deposited data. 2F_o–F_c (blue) and F_o–F_c (green/red) are contoured at the indicated levels. (C) Polder map around rigosertib contoured at 3.0 σ.