Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.

Abstract

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.

• Authors: Marco Jost, Yuwen Chen, Luke A Gilbert, Max A Horlbeck, Lenno Krenning, Grégory Menchon, Ankit Rai, Min Y Cho, Jacob J Stern, Andrea E Prota, Martin Kampmann, Anna Akhmanova, Michel O Steinmetz, Marvin E Tanenbaum, Jonathan S Weissman
• Journal: Molecular cell (Mol Cell) Vol. 68 Issue 1 Pg. 210-223.e6 (Oct 05 2017) ISSN: 1097-4164 [Electronic] United States
• PMID: 28985505 (Publication Type: Journal Article)
• Copyright: Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Chemical References

• Antineoplastic Agents
• KIF2C protein, human
• RNA, Guide
• Recombinant Fusion Proteins
• Small Molecule Libraries
• Sulfones
• Tubulin
• Tubulin Modulators
• Vinblastine
• ON 01910
• Kinesins
• Colchicine
• Glycine

Topics

• Antineoplastic Agents (chemistry, pharmacology)
• CRISPR-Cas Systems
• Colchicine (pharmacology)
• Drug Resistance, Neoplasm
• Gene Expression Regulation, Neoplastic
• Genetic Testing (methods)
• Genetic Vectors (chemistry, metabolism)
• Glycine (analogs & derivatives, chemistry, pharmacology)
• HeLa Cells
• Humans
• K562 Cells
• Kinesins (genetics, metabolism)
• Lentivirus (genetics, metabolism)
• Microtubules (drug effects, metabolism, ultrastructure)
• Mutation
• Myelodysplastic Syndromes (genetics, metabolism, pathology)
• RNA, Guide, Kinetoplastida (genetics, metabolism)
• Recombinant Fusion Proteins (genetics, metabolism)
• Small Molecule Libraries (pharmacology)
• Sulfones (chemistry, pharmacology)
• Tubulin (chemistry, genetics, metabolism)
• Tubulin Modulators (chemistry, pharmacology)
• Vinblastine (pharmacology)