Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061

Abstract 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.


10th Feb 2022 1st Editorial Decision
Thank you again for transferring your manuscript together with referee reports from a another journal to The EMBO Journal. Following our own assessment of the study and your responses to the previous reviews, as well as discussions with expert editorial advisors, we conclude that a study revised along the lines suggested in your tentative response letter would be of interest to our readership. I am therefore inviting you to resubmit a new version incorporating the already obtained data and planned experiments, as well the various other clarifications. In particular, it should be good to include results obtained with the mentioned new antibody for holoenzyme immunoprecipitation. In addition, please rewrite particularly the abstract and extend the background introduction as appropriate for a stand-alone publication. Please do not hesitate to contact me in order to discuss any specific points ahead of resubmission.
Detailed information on preparing, formatting and uploading a revised manuscript can be found below and in our Guide to Authors -adhering to these guidelines as closely as possible should greatly facilitate editorial processing at the resubmission stage.

Major new experimental data added to the revised manuscript.
We thank the reviewers for the comments provided to improve our manuscript. We have added the following major new data to our manuscript to strengthen our arguments and address concerns raised by the reviewers: 1) We have analysed if perphenazine (PPZ) interacts with PPP2R1A as originally claimed in Gutierrez et al., 2014. This is important to establish, as PPZ is the parent compound providing the starting point for iHAP1 development and mode of action of DT-061. The fact that PPZ can directly bind to PPP2R1A was newer shown by Gutierrez et al. (the authors just referred to a personal communication). Using ITC and NMR, we could not detect any binding of PPZ to PPP2R1A and we see no effect of PPZ in enzymatic assays (new data in Fig. 1, EV1 and Appendix S1-3). Our data thus question the mode of action of PPZ as claimed by Gutierrez et al. This is in line with our inability to detect direct effects of iHAP1 and DT-061 on PP2A complexes. 2) We have analysed the effect of DT-061 on stabilizing reconstituted PP2A-B56 using mass photometry (MP) measurements. Using the same concentrations of holoenzyme as in Leonard et al., we see no effect of DT-061 (new data in Fig. 1D). 3) We analysed the effect of DT-061 on the endogenous holoenzymes in HeLa and H358 cell lines using size exclusion chromatography of cell extracts, since we could not identify an antibody able to immunoprecipitate endogenous PPP2R1A. Using this approach, we see a clear co-migration of endogenous B56 with PPP2R1A and no sign of free B56 which would argue that there is no free B56 which DT-061 can act on. Furthermore, addition of DT-061 to cells and cell extract did not result in an increase in B56 co-migrating with PPP2R1A (new data in Appendix Fig. S6-7). 4) We have used an affinity tagged form of PPP2R1A to take a similar approach as in Morita et al. and Leonard et al. However, we did not detect an effect of PP2A-B56 composition by addition of DT-061 or iHAP1 (new data in Fig. 2C). 5) In an attempt to "mimic" the proposed mechanism of action of DT-061, we have used a stable cell line expressing inducible YFP-B56 We have previously shown that this YFP-B56 is functional, as it can suppress RNAi depletion of all B56 isoforms. After inducing YFP-B56 expression we see a large increase of PP2A-B56 holoenzyme formation by size-exclusion chromatography, yet no effect on cell growth. Under similar conditions DT-061 blocked cell growth (new data Fig. EV2B-C) 6) We have expanded our analysis of the cryo-EM structure reported in Leonard et al. and compared the tail of PP2AC to that of previous structures. All previous structures support the conclusion that the assignment of DT-061 is unambigious and can also be attributed to residues from the PP2AC tail (new analysis in Fig. EV2 and Appendix Fig. S8). 7) We show that the effect of iHAP1 on microtubules is not prevented by okadaic acid (we use it at concentrations that fully inactivate all PP2A complexes). This argues that the effect is not mediated by PP2A complexes (new data in Fig. 4) 8) We have expanded our analysis of the effect of DT-061 on ER and Golgi markers to H358 cells and have quantified all experiments. We have furthermore included okadaic acid treatments to determine if the effects we see with DT-061 are dependent on PP2A activity. The overall conclusion is that DT-061 affects Golgi and ER markers in both cell lines and largely independently of PP2A activity (new data in Fig. 6-7).

6th May 2022 1st Authors' Response to Reviewers 16th May 2022 1st Revision -Editorial Decision
Thank you for submitting your revised manuscript to The EMBO Journal. I have now carefully gone through your responses to the transferred original referee reports, and the changes made in response to my original decision letter. I am happy to say that we can now offer publication in our journal, as soon as a few remaining editorial points listed below have been addressed:

18th May 2022 2nd Authors' Response to Reviewers
The authors have made all requested editorial changes.

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