Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent
Authors: Yuwen Chen
Jacob J Stern
Martin Kampmann
Anna Akhmanova
Luke A Gilbert
Ankit Rai
Michel O Steinmetz
Marco Jost
Lenno Krenning
Grégory Menchon
Marvin E Tanenbaum
Andrea E Prota
Jonathan S Weissman
Min Y Cho
Max A Horlbeck
Journal: Molecular Cell (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: CRISPRa; CRISPRi; chemical genetics; drug mechanism of action; drug target identification
Affiliations: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA., Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA., Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, Department of Urology, University of California, San Francisco, San Francisco, CA 94158, USA., Hubrecht Institute - KNAW and University Medical Center Utrecht, 3584CT Utrecht, the Netherlands., Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland., Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3548CH Utrecht, the Netherlands., Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Chan-Zuckerberg Biohub, San Francisco, CA 94158, USA., Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland; Biozentrum, University of Basel, 4056 Basel, Switzerland., Hubrecht Institute - KNAW and University Medical Center Utrecht, 3584CT Utrecht, the Netherlands. Electronic address: ., Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: .

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. 

Retracted: No

PubPeer comments:
  Figure 5. TEM image of OPM coated emulsomes (arrows indicate coating). Increasing the contrast of Figure 5 reveals a curiously mosaic-like appearance, with the background formed from repeated copies of a small section. ![file] There is also evidence, when one increases the lightness to bring up details in the cores of the emulsome images, that these are repeated (sometimes with a 180-deg rotation) --although this is not so obvious in the coatings . ![file] , 
 Retracted [14 February 2022] . -------------------------------------- We, the Publisher and Editor of Journal of Liposome Research, have retracted the following article: Ajay Pal, Swati Gupta, Anil Jaiswal, Anuradha Dube Suresh P. Vyas (2012) Development and evaluation of tripalmitin emulsomes for the treatment of experimental visceral leishmaniasis, Journal of Liposome Research, 22:1, 62u201371, DOI: 10.3109 08982104.2011.592495 Following publication of this article, concerns about the integrity of Figure 5 were brought to the attention of the Editor and Publisher. The authors were contacted but could not provide the original image or sufficiently explain the irregularities in the image. The Editor has determined the findings of the article are unreliable and requested for the article to be retracted. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as u2018Retractedu2019. ,