TitleCombined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.
Publication TypeJournal Article
Year of Publication2017
AuthorsJost M, Chen Y, Gilbert LA, Horlbeck MA, Krenning L, Menchon G, Rai A, Cho MY, Stern JJ, Prota AE, Kampmann M, Akhmanova A, Steinmetz MO, Tanenbaum ME, Weissman JS
JournalMol Cell
Date Published2017 Oct 05
KeywordsAntineoplastic Agents, Colchicine, CRISPR-Cas Systems, Drug Resistance, Neoplasm, Gene Expression Regulation, Neoplastic, Genetic Testing, Genetic Vectors, Glycine, HeLa Cells, Humans, K562 Cells, Kinesin, Lentivirus, Microtubules, Mutation, Myelodysplastic Syndromes, Recombinant Fusion Proteins, RNA, Guide, Small Molecule Libraries, Sulfones, Tubulin, Tubulin Modulators, Vinblastine

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.

Alternate JournalMol. Cell
PubMed ID28985505
PubMed Central IDPMC5640507
Grant ListU01 CA168370 / CA / NCI NIH HHS / United States
K99 CA181494 / CA / NCI NIH HHS / United States
U01 CA217882 / CA / NCI NIH HHS / United States
R00 CA204602 / CA / NCI NIH HHS / United States
T32 GM007618 / GM / NIGMS NIH HHS / United States
F32 GM116331 / GM / NIGMS NIH HHS / United States
K99 CA204602 / CA / NCI NIH HHS / United States
T32 EB009383 / EB / NIBIB NIH HHS / United States
P50 GM102706 / GM / NIGMS NIH HHS / United States
R01 DA036858 / DA / NIDA NIH HHS / United States
/ / Howard Hughes Medical Institute / United States