TY - JOUR
T1 - Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent
AU - Jost, Marco
AU - Chen, Yuwen
AU - Gilbert, Luke A.
AU - Horlbeck, Max A.
AU - Krenning, Lenno
AU - Menchon, Grégory
AU - Rai, Ankit
AU - Cho, Min Y.
AU - Stern, Jacob J.
AU - Prota, Andrea E.
AU - Kampmann, Martin
AU - Akhmanova, Anna
AU - Steinmetz, Michel O.
AU - Tanenbaum, Marvin E.
AU - Weissman, Jonathan S
N1 - Funding Information:
We thank Eric Chow, Derek Bogdanoff (UCSF Center for Advanced Technology), Vincent Olieric, and Meitian Wang (Swiss Light Source) for technical assistance; Jeffrey Hussmann for help with data analysis; Marco Hein for sharing constructs; Kevan Shokat for pointing out rigosertib as a promising drug with a poorly characterized molecular target; Lynn McGregor (Shokat lab) for sharing ARS-853 and the H358 cell line; and Carol Gross and members of the Weissman lab for helpful discussions. This work was funded by the NIH (grants P50 GM102706 , U01 CA168370 , and R01 DA036858 to J.S.W. and post-doctoral fellowship F32 GM116331 to M.J.), the Swiss National Science Foundation (grant 31003A_166608 to M.O.S.), the European Research Council (starting grant ERC-STG 677936-RNAREG to M.E.T.), a fellowship from the Dutch Cancer Society (KWF) to M.E.T., NWO CW ECHO grant 711.015.005 to A.A., NIH/NCI Pathway to Independence Award K99 CA204602 to L.A.G., and an NIH/NCI Pathway to Independence Award ( K99 CA181494 ) and Stand Up To Cancer Innovative Research Grant to M.K. J.S.W. is a Howard Hughes Medical Institute Investigator. M.A.H., L.A.G., M.K., and J.S.W. have filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). M.E.T., L.A.G., and J.S.W. have filed a patent application for the SunTag technology (PCT/US2015/040439). J.S.W. is a founder of KSQ Therapeutics, a CRISPR functional genomics company. Oligonucleotide pools were provided courtesy of the Innovative Genomics Institute.
Publisher Copyright:
© 2017 The Author(s)
PY - 2017/10/5
Y1 - 2017/10/5
N2 - 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. 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.
AB - 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. 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.
KW - chemical genetics
KW - CRISPRa
KW - CRISPRi
KW - drug mechanism of action
KW - drug target identification
KW - genome-wide CRISPR screening
KW - microtubules
KW - rigosertib
UR - http://www.scopus.com/inward/record.url?scp=85030657644&partnerID=8YFLogxK
U2 - 10.1016/j.molcel.2017.09.012
DO - 10.1016/j.molcel.2017.09.012
M3 - Article
C2 - 28985505
AN - SCOPUS:85030657644
SN - 1097-2765
VL - 68
SP - 210-223.e6
JO - Molecular Cell
JF - Molecular Cell
IS - 1
ER -