Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2
Authors: A Thomas Look
Megan W Martel
Mark W Zimmerman
Adam D Durbin
Ken Morita
Radosław P Nowak
Nicole Prutsch
Nathanael S Gray
Shuning He
Cong Fu
Eric S Fischer
Jinhua Wang
Journal: Cell (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: MYBL2; PP2A; PPZ; cell cycle arrest; dopamine receptor

Abstract:  Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates. 

Retracted: No

PubPeer comments:
 Readers should become aware of a recent preprint entitled Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting that calls into question some of the conclusions obtained at Morita et al., 2020 paper. https: www.biorxiv.org content 10.1101 2021.07.08.451586v1 From the Abstract: The small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we establish the biological pathways targeted by these compounds. We find that iHAP1 directly targets microtubule assembly both in vitro and in vivo and thus works as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work argues that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology. , 
 Retracted [4 August 2022] . ----------------------------- This article has been retracted at the request of the authors. Their study showed that different PP2A holoenzymes, composed of distinct subunits and acting on unique substrates, can be specifically targeted by different small-molecule allosteric activators, including perphenazine. An important part of the study was the identification of a compound the authors called iHAP1 (improved heterocyclic activator of PP2A) that was suitable for in vivo studies in zebrafish and murine models because it did not interfere with dopamine signaling, which caused dose limiting off-target toxicity in the case of perphenazine. The authors also assessed iHAP1 for a second type of off-target activity involving inhibition of tubulin polymerization, and they reported that iHAP1 did not affect tubulin polymerization into microtubules (Figures S6D and S6E). Recently, Vit and coworkers published an article in The EMBO Journal showing that iHAP1 does in fact significantly inhibit tubulin polymerization (Vit et al., 2022, EMBO J. 41, e110611, https: doi.org 10.15252 embj.2022110611). The authors have subsequently used the same tubulin polymerization kit that they originally used for determining the effects of small molecules on the rate of tubulin polymerization and carefully optimized the conditions with the control compounds provided with the kit. After recalibration of the plate reader based on these controls, the authors determined that their original results are not reproducible and that iHAP1 does in fact potently inhibit tubulin polymerization. They are uncertain why their original analysis yielded inaccurate results. Unfortunately, the fact that iHAP1 inhibits tubulin polymerization renders uninterpretable their in vivo studies showing anti-cancer cell activity in zebrafish and murine models, because they cannot tell how much of the activity is due to activation of PP2A and how much is contributed by the anti-tubulin activities of this molecule. Because these in vivo studies and other in vitro studies in the paper prominently include iHAP1, all of the authors have agreed that the most appropriate course of action is to retract the paper. Many in vitro experiments in the paper that address different aspects of PP2A activation were performed with both iHAP1 and perphenazine, which does not possess detectable anti-tubulin activity. In view of the error involving iHAP1 and anti-tubulin polymerization, the authors are currently repeating each of these experiments in the original article in Cell; however, this will take a much longer time. They have decided the most responsible thing is to retract the paper now, so that others are not led to perform uninterpretable experiments with the iHAP1 compound. The authors regret and apologize for this mistake. , 
 See also https: retractionwatch.com 2022 08 10 doing-the-right-thing-harvard-researchers-retract-cell-paper-after-contradicting-work ,