Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061
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Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.
|Udgivet - 2022
Work at the Novo Nordisk Foundation Center for Protein Research is supported by grant NNF14CC0001. JN is supported by grants from the Danish Cancer Society (R269‐A15586‐B17), Independent Research Fund Denmark (8021‐00101B and 0134‐00199B) and Novo Nordisk Foundation (NNF18OC0053124 and NNF20OC0065098). The work was supported by Independent Research Fund Denmark (grant no. 7016‐00055) to NM and 6108–00542B to KM. JD is supported by the Novo Nordisk Foundation (grant agreement no. NNF18CC0033876). MW was supported by the Swiss National Fund (P2EZP3_178624) and the Danish Lundbeckfonden (2017‐3212). Work in the lab of MB is supported by grants from the Danish Cancer Society (R146‐A9322), the Lundbeck Foundation (R215‐2015‐4081) and the Novo Nordisk Foundation (NNF19OC0058504). This work is supported by grants R35GM119455 from NIH/NIGMS and R33CA225458 from NIH/NCI to ANK. AAJ acknowledges the Wellcome Trust for their support through a Senior Research Fellowship (202811) award. We thank Martina Barisic for technical assistance. Porcine brain for tubulin purification was kindly provided by Karsten P Hammelev, Copenhagen University, Denmark. NMR data were recorded at cOpenNMR, an infrastructure facility funded by the Novo Nordisk Foundation (NNF18OC0032996). We thank the NNF CPR protein production facility for help with expression and purification of PPP2R1A. We thank Magali Michaut and the Genomics Platform at reNEW/CPR for technical expertise, support and the use of instruments. The platform is supported by NNF17CC0027852. Data processing and analysis were performed using the DeiC National Life Science Supercomputer at DTU ( www.computerome.dk ). We thank Iain Cheeseman for providing the inducible CRISPR‐Cas9 cell lines and Michael Luckmann for compound structures. We thank Jukka Westermarck, Maja Koehn and Veerle Janssens for fruitful discussions. Stable cell lines or materials generated for this study can be requested by contacting Jakob Nilsson.
© 2022 The Authors. Published under the terms of the CC BY 4.0 license.
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