CoChat
Sign Up
Cited by 176Protein Tyrosine PhosphatasesProtein Kinase Regulation and GTPase SignalingSignaling Pathways in Disease

Selective PP2A enhancement through biased heterotrimer stabilization

Open access
Leonard, Daniel, Huang, Wei, Izadmehr, Sudeh, O’Connor, Caitlin M., Wiredja, Danica D., Wang, Zhizhi, Zaware, Nilesh, Chen, Yinghua +11 more·2020·Cell
DOI 10.1016/j.cell.2020.03.038PMC PMC7243596
Open this paper in CoChat — free accountOpen PDF

Abstract

Summary: Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory “B” subunits that compete for assembly and activation of heterogenous PP2A heterotrimers. Here we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-MYC. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

AI snapshot

Written from the paper’s full text.

TL;DR

DT-061 selectively stabilizes B56α-containing PP2A heterotrimers by binding a trimeric interface, driving dephosphorylation of substrates such as c-MYC and tumor suppression. A 3.6 Å cryo-EM structure and biochemical/cellular assays define this biased stabilization mechanism and suggest L309 methylation as a pharmacodynamic marker.

Key findings

  • The 3.6 Å cryo-EM structure of DT-061-bound AB56αC shows the ligand in an intersubunit pocket contacting all three PP2A subunits; it pins the C-tail near methylated L309, and without DT-061 the recombinant trimer dissociates during EM-grid preparation.
  • DT-061 increases B56α holoenzyme populations: NanoBiT complementation rises 3–6 fold at 1–2 hours, co-IP/mass spectrometry shows roughly two-fold more B56α trimers in xenograft tumors and cell culture, while B55α and PR72 remain unchanged; B56γ binding is unaffected, and a B56α T281K substitution abolishes stabilization.
  • DT-061 derivatives NZ-035, NZ-043, and NZ-044 fail to stabilize AB56αC and show reduced apoptosis/GI50 effects, validating the assigned binding pocket.
  • In vivo, methylated L309 increases at 1–3 hours and returns to baseline by 12 hours, c-MYC loss occurs at 1–3 hours and recovers by 6 hours, and TUNEL apoptosis tracks these changes; the recurrent R183W A-subunit mutation reduces B56α binding and L309 methylation, and DT-061 tumor suppression is absent in R183W tumors while dual kinase inhibitor treatment gives ~70–90% tumor inhibition across lines.

Methods

  • Recombinant PP2A A, C, and B56α subunits, including a T281K mutant, were expressed and purified; AB56αC was assembled and analyzed by size-exclusion chromatography, fluorescence polarization, surface plasmon resonance, and cryo-EM with molecular dynamics flexible fitting and refinement.
  • Cell-based work used H358 and H293T lines with NanoBiT/NanoBRET trimerization assays, V5-tagged A-subunit co-immunoprecipitation followed by mass spectrometry and SAINT scoring, MTT viability assays, immunoblotting, immunohistochemistry, and TUNEL staining.
  • In vivo studies used H358 xenografts in athymic nude male mice aged 6–8 weeks, with DT-061 at 5 mg/kg BID in a single-dose time course and a 29-day co-IP/interactome study, plus comparison to a dual kinase inhibitor combination.

Limitations

  • The paper does not include a dedicated limitations section; it raises open questions about refining SMAP nomenclature, how B56α expression predicts sensitivity or resistance, how these molecules behave when PP2A is dysregulated by inhibitors such as CIP2A, optimal timing for kinase-inhibitor combinations, and the need for atomic structures of each B-subunit isoform.
  • Evident from the text: in vivo efficacy and resistance findings rely heavily on H358 xenografts and one recurrent A-subunit mutation, and the structural work uses a recombinant trimer, so broader tumor contexts, patient-derived data, and cellular PTM complexity beyond L309 methylation are not fully captured.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2020.03.038

Cites

72 of 72 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 43 cited sources checked · 4 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Mutations at P179, R183, S256, and R258, which recur in disease, markedly alter PP2A holoenzyme composition in cells.

    “Interestingly, recurrent mutation of these residues (for example, P179, R183, S256, and R258) result in marked changes in the PP2A holoenzyme composition in cells (Haesen et al., 2016; O’Connor et al., 2019; Ruediger et al., 2011; Taylor et al., 2019).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Haesen et al., 2016 source
      Experimental — accuracy not yet measured
    • cites O’Connor et al., 2019

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Ruediger et al., 2011 source

      The Discussion (page not provided) identifies cancer-associated R183 mutations and reports that artificial R183 mutations impair binding to several PP2A regulatory subunits. It only predicts a similar effect for the cancer-associated variants. The cited paper does not establish that mutations at all four listed residues—P179, R183, S256, and R258—markedly change holoenzyme composition in cells. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Tau hyperphosphorylation in neurodegenerative disorders is directly linked to PP2A dysfunction, especially B55 holoenzyme dysfunction.

    “Structural and biological studies have shown that tau hyperphosphorylation in neurodegenerative disorders is directly tied to PP2A dysfunction, in particular B55 holoenzyme dysfunction (Sontag and Sontag, 2014; Wang et al., 1995; Xu et al., 2008).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Sontag and Sontag, 2014 source

      The section ‘Deregulation of PP2A/Bα: A Major Contributor to Phospho-Tau Pathology’ (page unavailable) says PP2A/Bα is the primary PP2A isoform that dephosphorylates tau and that its inhibition is associated with increased AD-like tau phosphorylation. The paper’s evidence centers on Alzheimer’s disease; it does not establish the asserted direct link across neurodegenerative disorders generally. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Wang et al., 1995 source

      The paper reports that PP-2A1 dephosphorylates hyperphosphorylated tau from Alzheimer paired helical filaments and helps restore its microtubule assembly-promoting activity (opening section; TABLE II; page numbers unavailable). It does not show that PP2A dysfunction causes tau hyperphosphorylation, identify B55 holoenzyme dysfunction, or establish the claim across neurodegenerative disorders. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Xu et al., 2008

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • Rests on an assumption: Neurodegenerative diseases such as Alzheimer's disease are characterized by hyperphosphorylation of tau.
  • Findingown data

    DT-061 treatment raises B56α-containing holoenzymes about two-fold in xenograft tumors relative to vehicle.

    “Co-IP data from DT-061 treated tumor samples demonstrated a significant increase, roughly two-fold, in B56α containing holoenzymes when compared to vehicle treated tumors (Fig. 1B).”
    Quote in the paperSubstantial claim
  • Findingown data

    DT-061's solvent-exposed phenoxazine part links C-subunit residues Y307 and P305 with A-subunit residues T102 and V103.

    “While the majority of DT-061 is buried inside the binding pocket, the solvent exposed phenoxazine moiety bridges between Y307 and P305 of the C subunit and T102 and V103 of the A subunit (Fig. 3A–D; Fig. S3).”
    Quote in the paperSubstantial claim
  • Findingown data

    DT-061's sulfonyl group makes electrostatic contacts with B-subunit K283.

    “The sulfonyl group forms electrostatic interactions with K283 of the B subunit.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 21
  • May combine two claims. 32

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CIP2A overexpression traps B56 family subunits, blocking B56-specific PP2A holoenzyme activity.

    “Alternatively, selective B subunit inhibition has also been characterized through the overexpression of endogenous PP2A inhibitors such as the Cancerous Inhibitor of PP2A (CIP2A) which selectively sequesters B56 family subunits to prevent B56 specific PP2A holoenzyme activity (Junttila et al., 2007; Wang et al., 2017).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Junttila et al., 2007 source

      The Results (page not provided) support that CIP2A inhibits c-Myc-associated PP2A activity, but not that it sequesters B56 subunits. The Discussion (page not provided) says CIP2A binds directly to c-Myc and that CIP2A depletion does not affect the PP2A–c-Myc interaction, contrary to the claimed sequestration mechanism. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites Wang et al., 2017 source

      The ‘CIP2A directly interacts with PP2A B56 tumor suppressor subunits’ section reports direct binding to B56α and B56γ, and the Figure 5 discussion proposes that a CIP2A dimer might trap B56 proteins. The Discussion treats binding to other B56 family members as a suspicion, not an established result. These passages do not establish that CIP2A overexpression sequesters the B56 family or prevents B56-specific PP2A holoenzyme activity. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingreasoning

    In standard culture, these derivatives caused no apoptosis, and in low serum their effect was much weaker by GI50.

    “In line with the inability to stabilize AB56αC heterotrimers, these derivative compounds did not induce apoptosis in standard cell culture conditions and had a significantly reduced effect in low serum culture conditions as determined by GI50s (Fig. 4C; Fig S4A) (Kauko et al., 2018; McClinch et al., 2018; Sangodkar et al., 2017a; Tohmé et al., 2019).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Kauko et al., 2018 source
    • cites McClinch et al., 2018 source

      The Results (page unavailable) report that TRC-766, an inactive compound structurally similar to SMAP, had negligible effects on cell viability and minimal to no effect on colony formation. They do not report an apoptosis test for TRC-766 or a low-serum GI50 comparison. The reported low-serum comparison concerns enhanced androgen-receptor degradation with SMAP, a different endpoint. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Sangodkar et al., 2017a source

      The Results report that the SMAPs tested in this paper induced cell death in cultured lung cancer cells; they do not identify those SMAPs as the derivative compounds in the citing sentence. The PP2A assay methods call TRC-766 inactive, but provide no apoptosis or low-serum GI50 result for it. Searches for derivative-compound apoptosis and low-serum GI50 comparisons found no passage addressing the attributed claim (page numbers unavailable). (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    SMAPs, small molecule PP2A activators derived from phenothiazines, cause dephosphorylation of chosen pathogenic targets like c-MYC and ERK and inhibit tumors in various in vivo cancer models.

    “A series of small molecule PP2A activators, previously termed SMAPs and engineered from phenothiazine parent compounds, drive dephosphorylation of select pathogenic substrates such as c-MYC and ERK with concordant tumor inhibition in an array of in vivo cancer models (Allen-Petersen et al., 2018; Kauko et al., 2018; Sangodkar et al., 2017b).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Allen-Petersen et al., 2018

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Kauko et al., 2018 source
    • cites Sangodkar et al., 2017b source

      The Discussion describes SMAPs as phenothiazine-derived PP2A activators, and the Results report reduced phosphorylated ERK and tumor growth in several in vivo models of KRAS-mutant lung cancer. Our copy does not report SMAP-induced dephosphorylation of c-MYC or demonstrate tumor inhibition across different cancer types. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PP2A is a major serine/threonine protein phosphatase family that is central to cellular homeostasis and is prone to dysregulation in human diseases.

    “The major serine/threonine protein phosphatase 2A (PP2A) family of phosphatases is central in maintaining cellular homeostasis and thus has a significant propensity for dysregulation in human diseases (Brautigan and Shenolikar, 2018; Junttila et al., 2007; Tsujio et al., 2005).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Brautigan and Shenolikar, 2018 source
    • cites Junttila et al., 2007 source

      The Discussion (page not provided) states that PP2A inhibition is required for malignant transformation and describes mechanisms of PP2A inhibition in human malignancies. This supports dysregulation in cancer, but the paper does not establish the broader claim that PP2A is central to cellular homeostasis or has a significant propensity for dysregulation across human diseases. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites Tsujio et al., 2005 source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    LCMT-1 adds and PME-1 removes a carboxyl methyl group on the C-tail's terminal leucine L309, a modification central to PP2A regulation.

    “Carboxyl methylation of the terminal leucine residue (L309) of the C-tail is carried out by Leucine Carboxyl Methyltransferase 1 (LCMT-1), removed by Protein Phosphatase Methylesterase 1 (PME-1), and is central in regulating PP2A biology (Jackson and Pallas, 2012; Ogris et al., 1999).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Jackson and Pallas, 2012 source
    • cites Ogris et al., 1999 source

      The Abstract reports that PP2A is reversibly methylated at its C terminus, that carboxymethylation affects PP2A activity, and that PME-1 demethylates the PP2A catalytic subunit. The passage on leucine 309 identifies it as the methylated terminal residue. Our copy does not identify LCMT-1 as the methyltransferase; the Abstract instead says the methyltransferase had been purified but not cloned. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Map validations were performed with PHENIX.

    “Map validations were performed using PHENIX (Adams et al., 2010).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Adams et al., 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The final atomic model was assessed with MolProbity.

    “The final atomic model was evaluated using MolProbity (Chen et al., 2010).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Chen et al., 2010 source

  • May combine two claims.

    Findingown data

    Without DT-061, the recombinant AB56αC trimer fell apart during EM-grid preparation, giving heterogeneous sub-complexes and mostly monomeric or dimeric 2D class averages.

    “Confirming our biochemical observations, in the absence of DT-061 the recombinant AB56αC trimer was unable to remain in its heterotrimeric composition during EM-grid preparations, resulting in heterogeneous sub-complexes with a majority of monomeric and dimeric two-dimensional class averages calculated from electron micrographs (Fig. 3E, F, blue box).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DT-061's trifluoromethoxy end contacts A-subunit residues E100 and E101 and B-subunit residue K316.

    “The trifluoromethoxy end of DT-061 reaches deep into a pocket to interact with E100 and E101 of the A subunit and K316 of the B subunit.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Reconstituted AB56αC stays together at micromolar levels but falls apart at nanomolar levels near 100 nM.

    “The sharp, symmetric peak of the chromatogram suggests that the reconstituted complex remains assembled at micromolar concentrations (Fig. 2A; Fig. S2AC). At nanomolar concentrations the complex dissociates, as indicated by the broadened SEC peak, which is indicative of a protein concentration dependent dissociation for the recombinant B56α complex that occurs around 100 nM (Fig. 2A, blue line).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The structure shows the C subunit bound to the A subunit's C-terminal region and the B subunit bound to its N-terminal region.

    “The C subunit anchors at the C-terminal region of the A subunit, while the B subunit interacts with the N-terminal region of the A subunit.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DT-061 roughly doubles B56α trimers in cell-culture co-IP samples, while B55α and PR72 levels stay the same.

    “In line with our observations from xenograft tumors treated with DT-061, B56α trimers specifically were enhanced in co-IP samples from cell culture, also roughly two-fold, whereas the population of B55α and PR72 remained unchanged (Fig. 1C, D; Fig. S1C, D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The C subunit's C-tail lies between the A and B subunits, right next to where DT-061 binds.

    “Importantly, the C-tail of the C subunit is sandwiched between the A and B subunits which is immediately adjacent to the DT-061 binding site (Fig. 3A–D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Between one and two hours of DT-061 exposure, NanoBiT luciferase complementation rises significantly by 3-6 fold, then returns to baseline by six hours.

    “Our data show a significant 3–6 fold increase in complementation of luciferase fragments between one and two hours of DT-061 exposure which is followed by a return to baseline by 6 hours after treatment (Fig. 1A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DT-061 occupies a distinct pocket at the junction between subunits, contacting residues from all three PP2A subunits.

    “DT-061 binds in a unique pocket formed at an intersubunit junction and interacts with residues from all three PP2A subunits (Fig. 3A–D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 3.6 Å cryo-EM structure reveals several intersubunit contacts, locates DT-061's previously unknown binding site, and explains how the trimer is stabilized.

    “Our 3.6 Å structure highlights multiple intersubunit interactions, identifies the previously unknown binding site of DT-061, and elucidates the molecular basis of trimer stabilization (Fig. 3; Fig. S3).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The DT-061-bound B56α-PP2A holoenzyme structure represents a fully assembled, active state.

  • May combine two claims.

    Findingown data

    DT-061 addition keeps the B56α complex stable and stops the holoenzyme from dissociating.

    “Under these same conditions, the addition of DT-061 stabilizes the B56α complex and prevents holoenzyme dissociation (Fig. 2A, red line).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PP2A-A R183W binds less L309-methylated C-subunit, but overall C-subunit binding and expression are unchanged.

    “Co-IP of isolated PP2A-A from H358 cells demonstrates that this mutation correlates with significantly less L309 methylated C-subunit bound, while having no effect on overall C-subunit binding or expression (Fig. 7A, B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: R183W is the most frequently recurring cancer-derived mutation in the PP2A gene family.

  • May combine two claims.

    Findingown data

    DT-061 suppressed EGFP and WT-A tumors but had no such effect in cells with the R183W mutant scaffold.

    “Whereas the dual kinase inhibitor treatment demonstrated ~70–90% tumor inhibition across all three lines, the tumor suppressive effects induced by DT-061 treatment in EGFP and WT-A tumors was absent in cells expressing the R183W mutant scaffold (Fig. 7D; Fig S7).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: R183W is the most frequently recurring cancer-derived mutation in the PP2A gene family.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The final cryo-EM 3D reconstruction reached 3.6 Å resolution according to the FSC 0.143 criterion.

    “yielding the final 3D reconstruction at 3.6 Å on the basis of Fourier shell correlation (FSC) 0.143 criterion (Rosenthal and Henderson, 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rosenthal and Henderson, 2003 source

  • May combine two claims.

    Findingown data

    Mass spectrometry of co-IPed scaffold from these tumors showed that resistant R183W-A cells had less PP2A-C L309 methylation and no B56α holoenzyme change after DT-061.

    “Additionally, mass spectrometry analysis of co-IP scaffold subunit from these tumors revealed that these resistant R183W-A expressing cells contained reduced levels of PP2A-C L309 methylation (Fig. 7E) and showed no change in B56α holoenzyme composition after treatment with DT-061 (Fig 7F).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: R183W is the most frequently recurring cancer-derived mutation in the PP2A gene family.

  • May combine two claims.

    Findingown data

    In the B56α T281K mutant, DT-061's stabilization and the increased B56α-AC affinity seen by SEC, FP, and SPR were entirely lost.

    “As expected, the stabilizing effect of DT-061 on the T281K mutant AB56αC heterotrimer and enhanced affinity of B56α for AC observed by SEC, FP, and SPR, was completely lost (Fig. 5D–F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    DT-061's contact with C-subunit Y307 holds the C-tail in the A-B cleft, probably making the assembled complex more stable.

    “Despite evidence of an inherently flexible C-tail in PP2A holoenzymes (Cho and Xu, 2007; Xu et al., 2006), the interaction formed between DT-061 and Y307 of the C subunit pins the C-tail in the cleft formed by the A and B subunits, which likely contributes to enhanced stability of the assembled complex.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cho and Xu, 2007 source
    • cites Xu et al., 2006

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    The designed DT-061 modifications support the assigned pocket and show that binding needs structural features found only in certain B-subunit-containing PP2A heterotrimers.

    “Thus, these strategic chemical modifications of DT-061 help to validate the assigned binding pocket of the active compound while highlighting the structural specificity required to accommodate binding, present only in specific B-subunit containing PP2A heterotrimers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    B subunit binding to the AC dimer and their substrate specificity depend on how accessible the C subunit's C-terminal tail is and on its post-translational modifications.

    “In particular, the capacity for B subunits to bind the core AC dimer, and thus direct substrate specificity, is dictated by the accessibility and PTMs of the carboxyl-terminus of the C subunit (C-tail) (Ikehara et al., 2007; Longin et al., 2007).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Ikehara et al., 2007 source
    • cites Longin et al., 2007 source

  • Classified as probably not a substantial claim.

    Premisecitation

    The regulatory B subunits of PP2A belong to four structurally distinct families (B55, B56, PR70/72, STRN) that share little to no sequence similarity.

    “The regulatory B subunits belong to four structurally distinct families (B55, B56, PR70/72, STRN) that exhibit little to no sequence similarity between them (Shi, 2009).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Shi, 2009 source

  • May combine two claims.

    Interpretationown data

    DT-061's selective enhancement of PP2A functions likely arises from regulation or biogenesis of PP2A holoenzymes, probably via more complex assembly.

    “These observations suggest that the selective PP2A functions enhanced by DT-061 are driven by PP2A holoenzyme regulation and/or biogenesis, presumably through an increase in PP2A complex assembly.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    AP-MS data were filtered and scored with SAINTexpress, and dot border intensity indicated statistical confidence with a 5% FDR cutoff.

    “SAINTexpress AP-MS software was used to filter and score AP-MS data from the tumor co-immunoprecipitants.”
    • Also stated in: § Quantification and Statistical Analysis · plain
      “the intensity of the dot border represents the statistical confidence in each value obtained (darker border equates to more confidence, FDR cutoff of 5%) (Chen and Gingras, 2007; Liu et al., 2011).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Chen and Gingras, 2007 source
    • cites Liu et al., 2011

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    PP6 offers a comparable framework for testing this therapeutic complex stabilization strategy.

    “and therefore provides a relatively similar framework to test this therapeutic complex stabilization strategy.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    That salt bridge fills the gap between A and B56γ, likely blocking the putative DT-061 pocket in assembled AB56γC.

    “This salt bridge occupies the space between the A and B56 γ subunits, and would occlude the putative DT-061 binding pocket in the assembled AB56γC complex.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Exploring cavities at protein interfaces can potentially modulate protein-protein assembly and adjust downstream functions.

    “These findings highlight the importance of exploring cavities at protein interfaces to potentially modulate protein-protein assembly, hence adjusting their downstream functions for both therapeutic and research-based applications.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    At the position corresponding to B56α 281, B56γ has a lysine instead of threonine; this lysine forms a salt bridge with A-subunit E101 in assembled AB56γC.

    “Notably, B56γ contains a lysine substituted for threonine at the analogous 281 position of B56α that forms a salt bridge with E101 of the A subunit in the assembled AB56γC PP2A heterotrimer (Fig. 5A–C) (Cho and Xu, 2007; Xu et al., 2006).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cho and Xu, 2007 source
    • cites Xu et al., 2006

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    In the DT-061 time-course study, tumors grown to 300 mm3 were randomized into control or DT-061 treatment groups.

    “For the DT-061 time-course analysis, tumors were grown to 300 mm3, then randomized into a control (DMA) 24 hour treatment group or a DT-061 (5 mg/kg BID) treatment group which included a 1 hour, 2 hour, 3 hour, 6 hour, 12 hour, and 24 hour group (7 total groups for randomization).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Each AP-MS experiment used three biological replicates, with fresh conjugated beads for each replicate.

    “Fresh conjugated beads were prepared for each biological replicate, three biological replicates were performed for each experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    IHC slides were randomized for blinded review.

    “Slides were sealed, and randomized for subsequent blinded review.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    TUNEL quantification used ImageJ cell counting, and ANOVA tested for differences in apoptosis among treatment groups.

    “Quantification was performed through the cell counter function on ImageJ (NIH). ANOVA test was performed to test for a significant difference between apoptosis among the treatment groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Two independent researchers scored mL309 staining extent using a composite score of intensity and percentage of positive cells.

    “Two independent researchers (D.L. and S.I.) scored specimens for extent of mL309 staining based on composite score based on intensity and percent of positive cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    DT-061 occupies a distinct heterotrimeric regulatory pocket, selectively stabilizing B56α-PP2A; this causes cell death and reduced tumor growth via B56α-directed dephosphorylation of substrates such as c-MYC.

    “We show that DT-061 binds a unique heterotrimeric regulatory pocket to selectively stabilize the B56α-PP2A holoenzyme thereby inducing cell death and attenuating tumor growth through B56α directed substrate dephosphorylation, including its most well characterized substrate, the oncogenic protein c-MYC (Allen-Petersen et al., 2018; Arnold and Sears, 2006; Farrell and Sears, 2014; Sheppard et al., 2”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Allen-Petersen et al., 2018

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Arnold and Sears, 2006 source
    • cites Farrell and Sears, 2014 source

  • May combine two claims.

    Interpretationown data

    The data support DT-061 stabilizing B56α-PP2A heterotrimers to cause the apoptotic effects and point to L309 methylation as a possible clinical biomarker.

    “These observations further support the notion that DT-061 stabilizes B56α-PP2A heterotrimers to drive the described apoptotic effects and highlights L309 methylation as a potential clinical biomarker.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Statistical analyses used GraphPad Prism 10, with unpaired two-tailed Student's t-tests and one- or two-way ANOVA plus Holm-Sidak or Bonferroni correction as appropriate.

    “Statistical analyses were performed by GraphPad Prism 10 software.”
    • Also stated in: § Quantification and Statistical Analysis · plain
      “Unpaired two tailed Student’s t-test and one or two-way ANOVA with post-hoc Holm-Sidak or Bonferroni correction were utilized as appropriate for multiple comparisons and indicated in the figure legends.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cell lines were tested monthly for mycoplasma contamination.

    “All cell lines underwent monthly testing for mycoplasma contamination (Lonza, LT07–710).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Constructs were verified by Sanger sequencing after cloning and mutagenesis.

    “After cloning and mutagenesis all constructs were sequence verified by Sanger sequencing.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Animal experiments were conducted in compliance with ethical regulations and with Case Western Reserve University IACUC approval.

    “All animal experiments and procedures were performed in compliance with ethical regulations and the approval of the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In a mouse xenograft model of lung adenocarcinoma, adding SMAPs to a dual kinase inhibitor combination (AZD6244/MK2206) yielded partial responses or complete tumor resolution in every treated animal, with statistically better outcomes than either agent alone.

    “combination of SMAPs with a dual kinase inhibitor cocktail (AZD6244/MK2206) in a mouse xenograft model of lung adenocarcinoma demonstrated either a partial response or complete tumor resolution in all treated animals, representing statistically better outcomes when compared to either treatment alone (Kauko et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kauko et al., 2018 source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For co-immunoprecipitation and interactome studies, V5-tagged PP2A-A H358 tumors grown to 100 mm3 were randomized to control or DT-061 treatment for 29 days.

    “For co-immunoprecipitation and interactome analysis studies, H358 tumors expressing V5-tagged PP2A-A scaffold subunit were harvested and injected as described above, then grown to 100 mm3, and randomized into a control (DMA) or DT-061 (5 mg/kg BID) treatment group, with treatment lasting 29 days.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Abnormal B56α holoenzyme activity is associated with cardiac remodeling dysfunction and arrhythmogenesis.

    “where aberrant B56α holoenzyme function is linked to dysfunction in cardiac remodeling and arrhythmogenesis (Bhasin et al., 2007; Kirchhefer et al., 2014).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Bhasin et al., 2007 source
    • cites Kirchhefer et al., 2014 source
46 of 46

  • The paper relies on this without support.

    R183W is the most frequently recurring cancer-derived mutation in the PP2A gene family.

    “An arginine to tryptophan substitution at position 183 (R183W) of the A subunit represents the most recurrent cancer derived mutation within the PP2A gene family.”

  • The paper relies on this without support.

    The DT-061-bound B56α-PP2A holoenzyme structure represents a fully assembled, active state.

    “Here we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-MYC.”

  • The paper relies on this without support.

    Neurodegenerative diseases such as Alzheimer's disease are characterized by hyperphosphorylation of tau.

    “Neurodegenerative diseases, such as Alzheimer’s disease, demonstrate pathognomonic hyperphosphorylation of tau.”

  • The paper relies on this without support.

    Hyperphosphorylated tau is pathogenic in neurodegenerative diseases.

    “driving dephosphorylation of pathogenic hyperphosphorylated tau in neurodegenerative diseases”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Retrospective studies of MGs have shown their power in inactivating enzymes, potentiating enzymatic activities, blocking protein-protein interaction, modulating client proteins, and modifying splicing.

    “Outside targeted protein degradation, retrospective studies of MGs have begun to showcase their versatile and promising power in inactivating enzymes by stabilizing autoinhibitory inter-domain interactions47, potentiating enzymatic activities by promoting protein oligomerization48,49, blocking protein–protein interaction by inducing unnatural homodimerization50, modulating the functions of client”

    From Defining molecular glues with a dual-nanobody cannabidiol sensor (2022) · cites this paper as [49]

  • PP2A dysregulation typically involves altered expressions of PP2A regulators and specific regulatory B subunits, while the catalytic and scaffolding subunits are less frequently affected.

    “These changes typically involve altered expressions of PP2A regulators and specific regulatory B subunits, while the catalytic (C) and scaffolding (A) subunits are less frequently affected [ 65 , 107 ].”

    From Targeting PP2A in cancer: an underrated option (2025) · cites this paper as [107]

  • Dysregulation of PP2A is a key driver of cancer development, most often arising from copy number variations, imbalances in regulatory proteins SET and CIP2A, or post-translational modifications rather than recurrent hotspot mutations.

    “Dysregulation of PP2A is a key driver of cancer development, most often arising from copy number variations (CNV), imbalances in regulatory proteins (SET and CIP2A), or PTMs rather than recurrent hotspot mutations.”

    From Targeting PP2A in cancer: an underrated option (2025) · cites this paper as [107]

  • Among PP2A subunits, the paralogs of the A subunit exhibit the highest frequency of mutations across cancer types, and these mutations are mainly recurrent, pathogenic, and frequently observed at the interface of the binding sites between the A and B subunits.

    “It should be noted that among the PP2A subunits, the paralogs of the A subunit exhibit the highest frequency of mutations across a variety of cancer types. These mutations are mainly recurrent, pathogenic, and frequently observed at the interface of the binding sites between the A and B subunits [ 107 ].”

    From Targeting PP2A in cancer: an underrated option (2025) · cites this paper as [107]

  • DT-061 stabilizes the fully assembled B56α-PP2A holoenzyme, keeping it in an active state.

    “The molecule DT-061 serves as a further activator of PP2A, which stabilizes the fully assembled B56α-PP2A holoenzyme, thus keeping it in an active state.”

    From Targeting PP2A in cancer: an underrated option (2025) · cites this paper as [107]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Time-course dosing is described as both single-dose and twice-daily

    Minor

    The results describe a single-dose time course, but the corresponding treatment group in the methods is specified as twice-daily dosing, including a 24-hour group.

  • NanoBRET label is described as both a luciferase component and a HaloTag

    Minor

    The Results call the label on B56α a complementing luciferase component, but the specified NanoBRET construct places a HaloTag at that end.

2 of 2

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1158/0008-5472.can-05-1103

    Info

    The cited work was retracted on 2024-03-15 (notice 10.1158/0008-5472.can-24-0434), not before this paper was published (2020-04).

    Cited work 10.1158/0008-5472.can-05-1103 · retracted 2024-03-15 · notice 10.1158/0008-5472.can-24-0434 · this paper published 2020-04

1 of 1

Cites

1 retracted

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Leonard, D., Huang, W., Izadmehr, S., O’Connor, C. M., Wiredja, D. D., Wang, Z., Zaware, N., Chen, Y., Schlatzer, D. M., Kiselar, J., Vasireddi, N., Schüchner, S., Perl, A. L., Galsky, M. D., Xu, W., Brautigan, D. L., Ogris, E., Taylor, D. J., & Narla, G.
Affiliations
  • Case Western Reserve University (US)
  • Tisch Cancer Institute (US)
  • University of Michigan (US)
  • University of Washington (US)
  • Icahn School of Medicine at Mount Sinai (US)
  • Max Perutz Labs (AT)
  • University of Virginia (US)
Corresponding author
  • Taylor, Derek J.
  • Narla, Goutham
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2020-04
Volume
181
Issue
3
Pages
688-701.e16
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2020.03.038
PMC
PMC7243596
OpenAlex
W3016570710
Cited by
176
References
72
Citations by year
2026: 19 · 2025: 29 · 2024: 28 · 2023: 32 · 2022: 27 · 2021: 30 · 2020: 11
FWCI
8.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Cancer Institute
  • American Heart Association
  • National Institutes of Health
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Signaling Pathways in Disease
Keywords
protein phosphatase 2A, protein phosphatase, enzyme complex assembly, small molecule inhibitors
MeSH terms
Amino Acid Sequence, Animals, Humans, Male, Mice, Nude, Models, Molecular, Enzyme Activators, Protein Subunits, Cell Line, Tumor, Multiprotein Complexes, Mice, Protein Phosphatase 2, HEK293 Cells, Heterografts

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read under definition version 3 (extract-v3) on 2026-09-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-09-28.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review