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Tumor Biology and Immunology

The Highly Recurrent PP2A Aa-Subunit MutationP179R Alters Protein Structure and Impairs PP2AEnzyme Function to Promote EndometrialTumorigenesisSarah E. Taylor1, Caitlin M. O'Connor2, Zhizhi Wang3, Guobo Shen3, Haichi Song4,Daniel Leonard1, Jaya Sangodkar5, Corinne LaVasseur6, Stefanie Avril1,7,Steven Waggoner8, Kristine Zanotti8, Amy J. Armstrong8, Christa Nagel8,Kimberly Resnick9, Sareena Singh10, Mark W. Jackson1,7,Wenqing Xu3, Shozeb Haider4,Analisa DiFeo11,12,13, and Goutham Narla5,13

Abstract

Somatic mutation of the protein phosphatase 2A (PP2A)Aa-subunit gene PPP2R1A is highly prevalent in high-gradeendometrial carcinoma.Thestructural,molecular, andbiologicalbasis by which the most recurrent endometrial carcinoma–specific mutation site P179 facilitates features of endometrialcarcinoma malignancy has yet to be fully determined. Here, weused a series of structural, biochemical, and biologicalapproaches to investigate the impact of the P179R missensemutation on PP2A function. Enhanced sampling moleculardynamics simulations showed that arginine-to-proline substitu-tion at the P179 residue changes the protein's stable conforma-tionprofile.A crystal structureof the tumor-derivedPP2Amutantrevealedmarked changes in A-subunit conformation. Binding tothe PP2A catalytic subunit was significantly impaired, disruptingholoenzyme formation and enzymatic activity. Cancer cells weredependent on PP2A disruption for sustained tumorigenic

potential, and restoration of wild-type Aa in a patient-derived P179R-mutant cell line restored enzyme functionand significantly attenuated tumorigenesis and metastasisin vivo. Furthermore, small molecule–mediated therapeuticreactivation of PP2A significantly inhibited tumorigenicityin vivo. These outcomes implicate PP2A functional inacti-vation as a critical component of high-grade endometrialcarcinoma disease pathogenesis. Moreover, they highlightPP2A reactivation as a potential therapeutic strategy forpatients who harbor P179R PPP2R1A mutations.

Significance: This study characterizes a highly recurrent,disease-specific PP2A PPP2R1A mutation as a driver ofendometrial carcinoma and a target for novel therapeuticdevelopment.

See related commentary by Haines and Huang, p. 4009

IntroductionUterine cancer is themost common gynecologic malignancy in

the United States with approximately 60,000 women diagnosedeach year (1). Although most cases of uterine endometrial carci-noma have favorable outcomes with recurrence-free long-termsurvival, outcomes for the high-grade, treatment-refractory histo-logic subtypes remain an important clinical problem (2, 3).Uterine serous endometrial carcinoma (USC) accounts for only10% of uterine cancer cases but a disproportionate 39% ofdeaths, with a 5-year survival of 55% (2). Uterine carcinosarcoma(UCS), a more aggressive mixed histologic subtype, is rare, <5%,but accounts for >15% of deaths, with a 5-year survival around35% (4–6). Importantly, despite an overall decline in cancer-related deaths in the United States, mortality rates for uterinecancers continue to rise (1). A lack of established disease drivingmechanisms for high-grade subtypes has limited the use oftargeted treatment strategies, an approach that has had somesuccess in the treatment of other cancers. New insight and vali-dation of biological drivers of disease progression are thereforeneeded to improve disease management. Large-scale genomicprofiling efforts have made substantial progress in identifyingalterations characteristic for USC and UCS (7–10). Foremost,both are typified by TP53 mutation, which is present in 80% to

1Department of Pathology, CaseWestern Reserve University School ofMedicine,Cleveland, Ohio. 2Department of Pharmacology, Case Western Reserve Univer-sity School of Medicine, Cleveland, Ohio. 3Department of Biological Structure,University ofWashington, Seattle, Washington. 4Department of Pharmaceuticaland Biological Chemistry, UCL School of Pharmacy, University College London,London, United Kingdom. 5Division of Genetic Medicine, Department of InternalMedicine, University ofMichigan, AnnArbor,Michigan. 6School ofMedicine, CaseWestern Reserve University, Cleveland, Ohio. 7Case Comprehensive CancerCenter, Case Western Reserve University, Cleveland, Ohio. 8Department ofObstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, Ohio.9Department of Obstetrics and Gynecology, MetroHealth, Cleveland, Ohio.10Department of Obstetrics and Gynecology, Aultman Hospital, Canton, Ohio.11Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor,Michigan. 12Department of Pathology, University of Michigan, Ann Arbor, Michi-gan. 13Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author:Goutham Narla, University of Michigan, 32299 SWood-land Road, Ann Arbor, MI 48106. Phone: 216-368-3111; Fax: 216-368-2968; E-mail:[email protected]

Cancer Res 2019;79:4242–57

doi: 10.1158/0008-5472.CAN-19-0218

�2019 American Association for Cancer Research.

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90% of cases. In addition to TP53, PPP2R1A mutation was alsomore frequent inhigh-grade subtypes, occurring in approximately30% of USC or UCS patients versus approximately 5% in patientswith the endometrioid subtype (UEC). PPP2R1A encodes thescaffold subunit of the protein phosphatase 2A (PP2A) tumor-suppressive phosphatase.

PP2A is a serine/threonine phosphatase that is involved in thephysiologic regulation of diverse signaling pathways. PP2A is alsoa tumor suppressor whose diminished activity contributes totransformation and tumor development (11–13). Although anumber of mechanisms can underlie diminished PP2A activityin cancer, notable is the occurrence of "hotspot" mutations toPPP2R1A, the gene encoding the PP2A Aa-subunit. The PP2Aholoenzyme is a heterotrimer composed of a scaffolding A-subunit (PP2A-A), catalytic C-subunit (PP2A-C), and regulatoryB-subunit (PP2A-B). Each is a subunit family composed of mul-tiple isoforms; for PP2A-A, this includes a and b isoforms.

The biogenesis of canonical PP2A heterotrimers is sequential:first a C-subunit binds to an A-subunit and is followed byincorporation of one of 15 identified B-subunits (14, 15). Severalactivation steps must also take place, facilitated by PP2A regula-tory proteins, to convert the C-subunit into its catalytically activeform and to modulate the binding of B-subunits (15). Critically,throughout this process, the A-subunit serves as a highly flexiblescaffolding protein to facilitate protein interactions and trimer-ization. Cancer-derived hotspot mutations of PPP2R1A cluster atthe structural interface between A- and B-subunits, and have beenshown to disrupt subunit binding to varying degrees (11, 16–18).A recent report characterizing several hotspot mutations foundthat these mutant proteins have increased binding to the PP2AinhibitorTIPRL, resulting inadominant-negativephenotype (18).Our data suggest this mechanism may be true for some hotspotmutations but may not hold true for others that are most specificand recurrent in high-grade endometrial carcinoma. Specifically,although PPP2R1A mutations are found in cancers of multipleorigins, they are striking for USC and UCS not only for their highprevalence, but also for the nearly exclusive occurrence of muta-tion at two hotspot sites: P179 and S256.

Our work focuses on P179R, the most common of theserecurrent, endometrial carcinoma–enrichedmutations, for whichwe have developed crystallography, modeling, and biochemicaldata to describe its impact on PP2A function. The P179Rmissensemutation induces global changes in A-subunit protein structureand its preferred conformational dynamics, which results in lossof interaction with the catalytic subunit and diminished holoen-zyme stability. When wild-type (WT) protein is restored in theP179R-mutant context, tumorigenesis is significantly inhibited.Moreover, pharmacologic activation of PP2A with a small-molecule activator of PP2A (SMAP) phenocopied this outcomeof tumor growth inhibition (19). Altogether, these works dem-onstrate that PP2A is a key tumor suppressor in endometrialcarcinoma and is functionally disrupted by recurrent P179RPPP2R1A mutation to drive tumorigenesis.

Materials and MethodsAnalysis of primary human endometrial and ovarian cancersamples

Deidentified tissue specimens were obtained from the CaseComprehensive Cancer Center (Case CCC) Gynecologic TumorBiobank (Dr. Analisa DiFeo). DNA and RNA were isolated from

tissue pieces using the AllPrep DNA/RNA Mini Kit (Qiagen) incombination with a QIAshredder (Qiagen) for initial tissuehomogenization. The DNA region of interest was amplified byPCR (PCR Master Mix, Promega) and submitted for Sangersequencing. Results were viewed using the software program4Peaks. DNA and RNA were stored at �20�C and �80�C,respectively.

Cell lines and culture conditionsThe UT42, UT89, UT150, and UT185 cell lines were generated

byDr. AnalisaDiFeo. These stable lineswere derived fromprimaryhuman endometrial cancer tissue following protocols describedpreviously (20). HEK293T cells were purchased from the ATCC.All lines were cultured in DMEM (Corning, 10-013) supplemen-ted with 10% FBS (GE Healthcare, SH30070.03) and 1% peni-cillin–streptomycin (GE Healthcare, SV30010). Cells were main-tained in humidified sterile incubator conditions with 5%CO2 at37�C. Cells are passaged once 70% to 90% confluency is reached,and are maintained in culture for no more than 20 passages.Freshly thawed cells are passaged 2 to 3 times before use inexperiments. Mycoplasma testing of cell lines is performed onceannually using adetection kit (Lonza, LT07-710), andpositive celllines are discarded. Authentication of cell lines was performed viashort tandem repeat profiling after the cell line was established(typically after two passages in culture), and compared with thatof the primary tumor.

Generation of stable cell linesStandard methodologies were used; details are provided in the

Supplementary Methods.

ImmunoblottingStandard methodologies were used; details and the primary

antibodies used are provided in the Supplementary Methods.

Real-time PCRStandard methodologies were used; details and primer

sequences are provided in the Supplementary Methods.

Coimmunoprecipitation and phosphatase activity assaysUT89 and UT42 isogenic cell lines were plated to achieve 70%

confluency at 24 (UT89) or 48 (UT42) hours. Cells were har-vested, and coimmunoprecipitation (IP) of V5-taggedproteinwasperformed according to the Dynabeads Co-ImmunoprecipitationKit (Invitrogen) protocol. Note that 1.5 mg Dynabeads per IPreaction were coupled to V5 antibody (Abcam ab27671) at aconcentration of 8 mg Ab/mg beads. Cells were lysed in a 1:9 ratioof mg cells to mL lysis buffer, and equal volumes of this pre-IPlysate were incubated with antibody-conjugated beads. Pre-IPlysates, and IP isolates obtained after elution from beads, werestored at �20�C for immunoblotting.

The phosphatase activity assay was performed using the IPprotocol with modifications: cells were lysed in a phosphataseassay buffer (25 mmol/L HEPES, 1 mmol/L MgCl, 0.1 mmol/LMnCl, 1%Triton-X, andprotease inhibitor); 5mgof protein lysatewas incubated with beads; beads were triple-washed with a washbuffer (25 mmol/L HEPES, 1 mmol/L MgCl, and 0.1 mmol/LMnCl); and aliquots were portioned out for immunoblotting oractivity assay. For assessment of phosphatase activity, beads withbound proteins were suspended in phosphatase assay buffer sup-plemented with 1 mmol/L DTT. Samples were incubated with

The PPP2R1A P179R Mutation Drives Endometrial Tumorigenesis

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increasing concentrations of DiFMUP (ThermoFisher, D6567) for15 minutes on a shaker at 37�C, at which point fluorescencewasmeasured at 365/455 nm. As a control for specificity of activity,50 nmol/L of okadaic acid (OA) was also added to select wellsof V5-WT IP isolate. Measurements from V5-EGFP IP isolateswere considered background and subtracted out. Phosphataseactivity rate was then calculated for V5-WT and V5-P179R asfluorescent units per minute incubation time. Final values werenormalized to total C-subunit protein in IP isolates as determinedby immunoblotting.

Cell-free GST pull-down assayApproximately 0.6 mmol/L of purified GST-B56a, 6 mmol/L of

His8-Ca, and 6 mmol/L of Aa WT or mutant protein wereincubated with 50 mL of GSH Sepharose 4B resin (GEHealthcare)for 1 hour at 4�C in binding buffer (20 mmol/L Tris, pH 8.0,150mmol/L NaCl, 5% glycerol, 0.05%Tween 20, 2mmol/L DTT;total volume of 250 mL). The beads were then washed withbinding buffer 3 times. The bound proteins were resolved onSDS-PAGE and stained with Coomassie Brilliant Blue. Theintensities of the bands were quantified by Image Lab Software(Bio-Rad), and values were normalized to the GST-B56a of WT.Each experiment was repeated 3 times.

Proteasome inhibition and half-life studiesCycloheximide solution was obtained from Sigma-Aldrich as a

stock solution of 100 mg/mL in DMSO and used at a concentra-tion of 100 mg/mL for experiments. Bortezomib (Velcade) wasobtained fromSelleckChemicals; a stock solutionwasprepared inDMSO and diluted to 1 mmol/L for experiments. Treatments wereadded 24 hours after cell plating. At the reported timepoints, cellswere harvested and lysed for protein immunoblotting. To calcu-late protein half-life, cycloheximide protein quantification datawere Ln-transformed andfitwith a regression lineusingGraphPadPrism 7 software. Details of the statistical analysis of these dataare presented in the Supplementary Methods. The slope of thebest-fit line was used to calculate half-life with the formulaT(1/2) ¼ ln(2)/slope.

Clonogenicity assayStandard methodologies were used; details are provided in the

Supplementary Methods.

SMAP dose–response assayEvaluation of SMAP treatment–induced growth inhibition was

performed to determine the IC50 of four endometrial carcinomacell lines. Cells were plated into 96-well plates at optimized celldensities such that approximately 90% confluency is achieved at72 hours. Twenty-four hours after plating, wells received mediacontaining DMSO or SMAP-061, with triplicate wells per treat-ment condition. The IncuCyte live-cell imaging system (EssenBiosciences) was utilized to monitor cell number in each well for72 hours. The fold change in cell number was determined forDMSO or SMAP, at each dose, and used to construct the dose–response curves. In GraphPad Prism, data were log-transformedand fit with a nonlinear curve.

Xenograft tumor assaysAll animal work was conducted in accordance with Case

Western Reserve University Animal Resource Center–approvedprotocols and ethical guidelines. For the subcutaneous xenograft

studies, 1�106 (UT89)or 5�106 (UT42) cells suspended in a 1:1mixture ofmatrigel, and cell media were injected s.c. into the rightflank of 6- to 8-week-old female Balb/c nu/nu mice. Tumorgrowth was assessed by caliper measurement, and volume (V)was calculated using the formula V ¼ 0.5 � (L �W2), where L ¼length andW ¼ width. At study end, tumor tissue was harvested,formalin-fixed, and paraffin-embedded for histologic evaluation,or snap-frozen in liquid nitrogen for protein isolation andimmunoblotting.

For the UT42 intrauterine xenograft study, procedures weremodeled after a published study (21). In brief, 6- to 8-week-oldfemale athymic NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)were put under general anesthesia, a small abdominal incisionwas made, and the left uterine horn was identified and ligated ateither end using 4-0 Vicryl suture (Ethicon). Note that 5 � 106

cells suspended in 50 mL sterile PBSwere then injected into the leftuterine horn; uterine enlargement and absence of visible leakagewere confirmed. Injections were performed across 3 days for anequal number of animals per experimental group. The study wasended at 8 weeks from the date of injection. Animals wereeuthanized, and the left and right uterine horns were isolated,evaluated for tumor presence, and weighed intact for all animals.Metastatic nodules throughout the abdominal cavity were col-lected, counted, and weighed separately from the uterus as the"metastatic tumor burden." Collected tissue specimens wereformalin-fixed andparaffin-embedded for hematoxylin and eosin(H&E) staining. Upon microscopic evaluation for tumor pres-ence, the largest linear dimension (LLD)was calculated as the sumof the lengths (in mm) of all tumor cell foci observed in a singlesection. Metastases were also confirmed microscopically.

The UT42 SMAP treatment study was performed using patient-derived xenograft (PDX). Tumor pieces were surgically implantedinto the flank of female NSG mice. Successful outgrowth wasmonitored, and upon reaching a tumor volume of 100 mm3,animals were enrolled and randomly assigned to a treatmentgroup. Treatment was delivered by oral gavage once daily:vehicle control (N,N-Dimethylacetamide, DMA), 50 mg/kgSMAP-1154, or 100 mg/kg SMAP-1154. Animal body weightand tumor volume were measured every 2 to 3 days for thestudy duration. Animals were observed for signs of toxicity(i.e., debilitating diarrhea, abdominal stiffness, jaundice,hunched posture, and rapid weight loss). Animals were eutha-nized once morbidity criteria were met as established in theprotocol; in all instances, this was due to a tumor size thataccounted for >10% of body weight. Terminal sacrifice wasconsidered the survival endpoint for construction of Kaplan–Meier curves. Tumor tissue was harvested 2 hours after a finaltreatment dose and was formalin-fixed for IHC evaluation orsnap-frozen in liquid nitrogen for immunoblotting.

H&E staining was performed following standard protocols.H&E-stained sections were imaged using a Leica SCN400 slidescanner at 20x magnification. Images were exported through theSCN400Digital Imaging Hub and cropped where appropriate forpublication (no other image adjustments were performed).

Protein expression, purification, crystallization, and structuredetermination

A mouse PP2A Aa P179R–mutant construct was clonedusing site-directed mutagenesis based on a mouse WT PP2A AapGEX-4T1 construct with an N-terminal GST tag and a tobaccoetch virus (TEV) protease cleavage site in between (22). The GST

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Cancer Res; 79(16) August 15, 2019 Cancer Research4244




fusion protein was overexpressed in Escherichia coli BL21 (DE3)cells grown in Luria brothmedia. Bacteria cell pelletswere lysedbysonication. TheGST fusionproteinswere eluted fromGlutathioneSepharose 4B beads. GST tag was removed by TEV at 4�C over-night. Then the proteins were further purified by an anionexchange column, and finally purified by gel filtration on aSuperdex 200 10/300 GL column (GE Healthcare). The peakfractions were pooled and concentrated to approximately 5 mg/-mL in a buffer containing 10mmol/L Tris HCl, pH80, 100mmol/L NaCl, and 2 mmol/L DTT.

The hanging-drop vapor diffusion method for crystallizationwas used to prepare crystals of mouse PP2A Aa P179R. To obtainprotein crystals for structural study, 1 mL of protein sample(5 mg/mL) was mixed with 1 mL of well solution containing0.2 mol/L sodium malonate, pH 7.0, and 20% w/v PEG 3,350.The crystals were flash frozen by liquid nitrogen in a cryogenicsolution containing 0.2 mol/L sodium malonate, pH 7.0, 10%glycerol, and 20% w/v PEG 3,350.

Screening and data collection were performed at the AdvancedLight Source, beamline 8.2.1 at wavelength 1.00 Å. Details of thedata collection and refinement are provided in the SupplementaryMethods. All diffraction data were processed by HKL2000 (23).The initial phase was determined by molecular replacement inPhaser with model from PDB: 2IAE (22, 24). All models wereimproved using iterative cycles of manual rebuilding with theprogram COOT and refinement with Refmac5 of the CCP4 6.1.2program suite (25, 26).

Molecular dynamics simulationsStructural coordinates for PP2A Aa were retrieved from the

Protein Data Bank (PDB: 2IAE; ref. 22). The mutants were con-structed in silico using the ICM mutagenesis program (27). TheAmber ff14SB force field was used to describe the systems, withexplicit TIP3P water molecules (28–30). PROPKA 3.1 andPDB2PQR 2.1, as implemented in the proteinPrepare() function-ality of HTMD, were used to determine the protonation state ofthe residues (31). Note that 1,000 steps of steepest descent wereused to minimize all systems. Energy minimization and equilib-rium for volume relaxation were carried out under NPT condi-tions at 1 atm, with initial velocities sampled from the Boltzmanndistribution at 300 K. The temperature was kept at 300 K by aLangevin thermostat. The PME algorithm was used for electro-static interactions with a cutoff of 1.2 nm. A 1.05 ms productionrun was carried out for all the systems in the NVT ensemble withhydrogen mass repartitioning that allowed for a time step of 4 fs.All simulations were performed using the ACEMD program usingan identical protocol (continued in the Supplementary Methods;refs. 32–38).

Statistical analysisResults are displayed asmean� SD, unless otherwise indicated.

In each figure legend, n indicates the number of independentexperimental replicates completed or, when in reference to anin vivo study, the number of animals per group. Thesewere distinctsamples whose data points were averaged to obtain groupmeans.The statistical tests used are identified in the figure legends and/ortext. All Student t tests performed were two-sided. GraphPadPrism7 softwarewas utilized for graph construction and statisticalanalysis.

Data availabilityThe atomic coordinate and structure factor of mouse PP2A Aa

P179R is deposited in the PDB with the accession code 6EF4.

Other data that support the findings of this study are availablefrom the corresponding author upon reasonable request.

Code availabilityDetails regarding the application and use of previously pub-

lished algorithms were available upon request.

ResultsMutation of PPP2R1A at P179 and S256 is specific tohigh-grade subtypes of endometrial carcinoma

Although three amino acid residues are notable PPP2R1A hot-spot mutation sites in cancer (Fig. 1A), two of these residues, P179and S256, display a striking specificity in occurrence that is incontrast to the third, most frequently mutated site, R183 (Fig. 1B).Specifically, P179 and S256 mutations are highly enriched inendometrial carcinoma, occurring nearly exclusively in USC andUCS, and also accounting for nearly all PPP2R1A mutations inthese endometrial carcinoma subtypes (Fig. 1B and C). This is instark contrast to the R183mutation, whichwas identified in cancersof >20 different organ systems, with colorectal carcinoma being themost common. Although R183mutations do occur in endometrialcarcinoma, they are much more prevalent in the less aggressiveendometrioid carcinoma (UEC) subtype. Critically, this differentialpattern of occurrence may indicate differing mechanistic rolesthrough which these mutations contribute to cancer. In addition,mutation at these hotspot sites is also largely mutually exclusive.Across cancer, only one tumor was found to harbor two PPP2R1Ahotspot mutations: a mixed ductal and lobular breast carcinomawas identified with both R183W and S256F mutations. In endo-metrial carcinoma, occasional tumors were found to harbor morethan one PPP2R1Amutation but in none of these cases were theretwo co-occurring hotspot site mutations (Supplementary Fig. S1A).

The P179 and S256 mutation sites reside in HEAT domains 5and 7, respectively, of the PP2AAa-subunit. The P179mutation isthe more common of the two and is substituted with arginine(P179R, 48 cases), lysine (P179L, 2 cases), or threonine (P179T, 1case). The S256 is mutated to phenylalanine (S256F, 15 cases) ortyrosine (S256Y, 6 cases). Importantly, large-scale sequencingstudies have revealed that 28% to 32% of USC patients and8% to 12% of UCS patients harbor one of these mutations (TheCancer Genome Atlas, MSK-IMPACT; Supplementary Fig. S1A).These patient cases display features characteristic of thehigh-gradeUSCandUCS subtypes inwhich theyoccur, andwhich are distinctfrom, and often in contrast to, the characteristic features of UECtumors. First, P179-mutant tumors are frequently high-stage, withgreater than 50% of patients diagnosed with stage III or IV disease(Supplementary Fig. S1B). A stage III or IV diagnosis indicatescancer progression involving extrauterine dissemination. Thisadvanced disease is typical of USC and UCS, whereas 80% ormore of UEC tumors are low-stage. Correspondingly, P179tumors, similar to all USC tumors, display poor disease-freesurvival (Supplementary Fig. S1C). At the genome level, P179tumors present with other defining features ofUSC andUCS, suchas a low overall mutational burden, high copy-number alteration,TP53 mutant, and MYC, CCNE1, or ERBB2 amplification (Sup-plementary Fig. S1D–S1F). Hallmarks that distinguish UEC,including microsatellite instability (MSI) or PTEN, ARID1A, orCTNNB1 mutation, are rare in P179 tumors.

We sought to identify patient tumor specimens harboringrecurrent PPP2R1Amutations in the Case CCCGynecologic Tumor






Biobank. Seventy-four endometrial carcinoma tumors and 18 ovar-ian carcinoma tumors underwent targeted Sanger sequencing atthe HEAT 5 and 7 hotspot foci. Sixteen percent (15 tumors) werefound to harbor PPP2R1Amutations, and allwere at residues P179,R183, or S256 (Supplementary Fig. S2A and S2B). In addition to

the previously reported P179R and P179L variants, we also identi-fied a new variant, P179H, in a mixed serous-/endometrioid-subtype tumor (Supplementary Fig. S2B and S2C). In this cohort,38% of USC tumors were found to harbor P179/S256 site muta-tions (Supplementary Fig. S2A). Consistent with previous studies,

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Figure 1.

P179R/L/T and S256F/Y Aamutations are highly specific to high-grade endometrial cancer subtypes. A, The distribution of PPP2R1Amutations identified inhuman cancers across the Aa-subunit protein length (adapted from cBioportal.org; refs. 64, 65). Inset, image of a PP2A heterotrimer (PDB: 2IAE) indicating thelocation of mutated amino acids at the A-/B-subunit interface. B, The cancers in which point mutation of the P179, S256, or R183 residue was identified. C,Occurrence and distribution of PPP2R1Amutations in endometrioid (UEC) and serous (USC) subtypes of endometrial cancer. ACYC, adenoid cystic carcinoma;AD, adenocarcinoma; CCOV, clear cell ovarian carcinoma; HGSOC, high-grade serous ovarian carcinoma; IDC, invasive ductal carcinoma; MDLC, mixed ductal andlobular carcinoma; UCCC, uterine clear cell carcinoma; UUC, uterine undifferentiated carcinoma.

Taylor et al.





our Sanger sequencing results demonstrated that these tumors arefrequently heterozygous for mutation, with a WT allele remaining(Supplementary Fig. S2C; refs. 39–41). However, for a subset of tu-mors, there was no WT allele detected, which suggests that muta-tions can be either homozygous or co-occur with allelic deletion.

P179R-Aa disrupts PP2A holoenzyme assemblyStudies have previously reported altered interactome and holo-

enzyme complex formation with cancer-associated mutant iso-forms of the PP2A Aa-subunit (16–18). However, these studieswere not performed in USC or UCS endometrial carcinoma cells,which is the most relevant disease context for studying the P179and S256 mutations. Thus, we generated a primary serous endo-metrial carcinoma cell line, UT89, to characterize the interactomeof P179R, which represents the most common missense mutationin high-grade endometrial carcinoma. For comparison, we alsoevaluated S256F and R183W. Cell lines with stable expression ofV5-tagged WT or mutant Aa-subunit were generated using a lenti-virus approach, and tagged Aa protein was coimmunoprecipitatedwith its binding partners from whole-cell lysates. Binding to PP2Afamily members was evaluated by Western blotting of coimmu-noprecipitants. Overall, the P179R mutation induced significantdisruption of PP2A holoenzyme formation, presenting with asignificant reduction in binding to members of each B-subunitfamily except the Striatins, for which binding remained intact, aswell as a significant loss of catalytic C-subunit binding (Fig. 2A–C).By comparison, S256F and R183W mutants also demonstratedlosses in B-subunit interactions, but in many cases, these losseswere less severe. Interestingly, P179R and S256F shared a near-complete loss of binding to the B55a-subunit, whereas R183Wretained some binding. Importantly, P179R was the most severelyimpaired in its interaction with the PP2A catalytic C-subunit.Finally, a published report identifies increased TIPRL binding tomutant Aa as a dominant-negative mechanism through whichPP2A is inactivated in cancer (18). We therefore also assessedmutant protein binding to TIPRL. From our results, the P179Rand S256F mutants do not display increased TIPRL binding in aserous endometrial carcinoma cell line (Fig. 2A). Meanwhile,R183W did display this gain of binding.

To complement the co-IP approach, we additionally employeda cell-free binding assay to evaluate the impact of P179 mutationon holoenzyme assembly (Supplementary Fig. S3A and S3B).GST-tagged B56a-subunit was incubated with purified C-subunitandpurifiedWTormutant Aa-subunit proteins, and then isolatedand assessed for binding. When in the presence of P179 mutantAa isoforms, assembly of the complete Aa-B56a-C holoenzymeheterotrimer was impaired. This is notable, as even the sole loss ofB56a holoenzymes has been identified as sufficient for celltransformation when substituted into an established transforma-tion model involving PP2A inactivation (13).

Altogether, these data support the previous literature describingloss of PP2A subunit interactions upon Aa mutation (16–18).Moreover, it highlights a near-complete loss of catalytic subunitbinding to P179R-Aa, which ultimately suggests amarked changein protein structure that influences C-subunit contact pointsdistant from the site of mutation.

The P179R-Aa protein displays altered conformationaldynamics

To understand how the overall protein structuremay be alteredby the P179R mutation, we determined the crystal structure of

the P179R-mutant protein at 3.4Å resolution (Fig. 2D and E).Comparison of this PP2A P179R-Aa structure with the WT-Aacrystal structure (PDB: 1B3U) revealed an obvious conformation-al difference between themutant andWT proteins (Fig. 2D and E,overlays; ref. 42). Although we cannot rule out a contributionfrom crystal packing, our crystal structure indicates a change of theconformation-energy landscape of the PP2A P179R-Aa protein.

Therefore, we next performed classical and enhanced samplingmolecular dynamics simulations of WT-Aa and P179R-Aa toidentify stable protein conformations. Free energy landscape(FES) plots were generated as a function of j and y dihedralangles of theP179andmutantR179 residues, for both apoAa andAa in complex with the C-subunit (Fig. 3A and B; SupplementaryFig. S4A–S4P). These landscapes provide evidence that the R179residue of the Aamutant is capable of exploring an altered FES toadopt multiple metastable conformations of the Aa-subunitprotein, which may alter interactions with other PP2A subunitsand proteins. We further extracted and analyzed the most stableconformations from the free energy minima of the WT (basin A)and mutant (basin C) FES (Fig. 3A and B). Importantly, when weextrapolate the obtained P179R-Aa crystal structure on the FES ofthe simulated P179R-Aa, we found that the conformationadopted by the crystal structure is indeed one of the stableconformations and can be found in the largest minimum of theR179 apo Aa FES (Fig. 3B, indicated by star).

Due to a cyclized side chain, P179 does not make interresidueinteractions in theWT crystalline conformation or throughout thecourse of any of ourmolecular dynamics simulations. By contrast,the R179 residue of the mutant protein makes direct interactionswith nearby residues. In the resolved crystal structure, the guani-dinium side chain of R179 orients toward the solvent, but stillinteracts with the side chain of Q217 of the adjacent HEAT repeat(Fig. 3C, white structure). Comparably, extracting themost stable,representative conformation from the largest basin on the FESidentifies an ion pair interaction betweenR179 and E216 (Fig. 3C,cyan structure). In both, the interaction of R179 is facilitated by anadditional ion pair interaction between R182 and D215.

The cyclic side chain of proline is effective in imparting con-formational rigidity to the secondary structure of proteins (43).Proline is also exceptional in the cis-trans isomerization ofthe peptidyl-prolyl backbone dihedral angle (w; defined as Ca-C-N-Ca) preceding itself (0� for cis and �180� for trans). The cis-trans configuration is isoenergetic, with the difference beingapproximately 1 kcal/mol between the two states (44–46). Thisisomerization is an important structural mechanism throughwhich proteins achieve large conformational changes and reachvarious macrostates of multidomain proteins without affectingthe covalent structures. Therefore,mutationof proline canhave aninfluence not only on the conformation of the protein but also onthese biological processes (45–48). To study the structural influ-ence of P179Rmutation on isomerization of the Aa P179 residue,including its dynamics in binding to the catalytic C-subunit, wecarried out additional simulations of theWT andmutant residues.First, we analyzed cis-trans state as a function ofwdihedral of P179(Ca178-C178-N179-Ca179) in both Aa/C complex and apo statesof Aa. The simulations identified that the w dihedral of theWT P179 residue exists predominantly in the trans configuration(Supplementary Fig. S4Q and S4R). This is more pronounced inthe apo statewhen comparedwith the complex. By contrast, in theP179R mutant, the w dihedral of R179 preferentially adopts a cisconfiguration in apo and complex states (Supplementary Fig. S4S






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P179R-Aa displays impaired binding to both PP2A B- and C-subunits, which is supported by a global protein conformation change in the resolved crystalstructure. A,Western blots of co-IP isolates reveal altered interactomewith mutant P179R-Aa protein. B and C,Quantification of the percent C- or B-subunitbinding relative toWT. Band intensity measurements for subunit protein in the co-IP samples were normalized to the amount of subunit protein present in thepre-IP input. Data are the average of three independent co-IP experiments (n¼ 3) and represent the mean� SD. Statistical significance was determined by theStudent t test (P179R vs. WT). � , P� 0.05; �� , P� 0.01; ��, P� 0.001; n.s., not significant. D and E,Overlay of the resolved P179R-Aa crystal structure (green;PDB: 6EF4) with a publishedWT-Aa crystal structure (orange; PDB: 1B3U; ref. 42). Crystallographic data collection and refinement details are provided in theSupplementary Methods.

Taylor et al.





Figure 3.

Molecular modeling reveals distinct conformational dynamics of the P179R-Aa–mutant protein.A and B, FES plots of the apo Aa-subunit (top) and Aa/Ccomplex (bottom) generated as a function of j andy dihedral angles of residues P179 and R179. A, The P179WT residue explores a similar conformationallandscape in both apo and complex states. B, By comparison, the R179-mutant FES is modified, indicating sampling of additional conformations. The landscapebetween apo and complex states of R179 is also altered, highlighting additional sampling of interactions when in the Aa/C complex. Labeled basins representenergy minima, with basins A and C representing the most populated minima for P179 and R179, respectively. The resolved crystal structure (Fig. 2D and E)extrapolates onto basin C of R179, indicated by the star. C and D, A comparison of interresidue interactions made by R179. C,Overlay of the P179R-Aa crystalstructure (white), with the representative conformation extracted from the largest populated basin of the apo R179 Aa FES (cyan). In the crystal structure, R179makes ion pair interactions with Q217, while R182 interacts with D215. In the simulated apo P179R-Aa, R179 interacts with E216, whereas the interaction betweenR182 and D215 remains unaltered. D, In the representative conformation of P179R-Aa in the Aa/C complex, R179 makes an ion pair with D215, whereas R182interacts with the side chain of N211. E and F, Conformational changes observed in the representative structures extracted from the most populatedminima fortheWT Aa/C complex (E) and the P179R-mutant Aa/C complex (F).






and S4T). In this cis conformation, R179 makes a strong ion pairwith D215, whereas R182 interacts with N211 (Fig. 3D).

Next, we analyzed the global conformational changes and theeffect of mutation on C-subunit binding to the Aa-subunit. TheFES plots indicated a complete change in conformational land-scape between the WT and mutant complex (Fig. 3A and B). Weextracted the representative conformations from the most popu-lated minimum of the Aa/C complex FES (basin A in P179 WTand C in R179mutant). TheWT-Aa-subunit adopts a "clam-like"conformation, with the catalytic subunit sandwiched between thetwo ends of the Aa-subunit (Fig. 3E). The catalytic subunit isdeeply embedded within the ends, thereby maximizing its inter-actionswith theAa-subunit. In theP179Rmutant, theAa-subunitprefers to adopt a near-closed "donut-shaped" conformation(Fig. 3F). The result is that the C-subunit appears to be squeezedout of the complex and now makes peripheral interactions withthe Aa-subunit, which is indicative of a destabilized complex. Wecalculated the binding energy for theC-subunit to either theWTorP179R mutant Aa-subunit, and determined them to be 236 and347 kJ/mol, respectively. The greater binding energy betweenP179R-Aa and C, versus WT-Aa and C, is indicative of a greaterC-subunit binding affinity for the WT isoform than the mutantisoform.This is again consistentwithadestabilizedcomplex and insupport of observations from the extracted structures. Altogether,these data show that a P179Rmutation of the Aa protein leads toconformational disruption and a destabilized interaction with theC-subunit, which in turn lends context to the altered P179R-mutant interactome described previously.

Impaired binding to P179R-Aa results in C-subunitdestabilization

Expression of the P179R-mutant isoform in UT89 cells wasfound to reduce total protein levels of C- and B-subunits to adegree that reflected their respective loss of binding in co-IPexperiments (Fig. 4A). Indeed, the loss of catalytic C-subunit totalprotein, as well as the regulatory B55a- and B56a-subunit pro-teins, was significant for the P179R mutant, which had demon-strated marked impairment of binding and conformation-relateddestabilization of the Aa/C complex (Fig. 4B). This led us tohypothesize that impaired subunit incorporation into the trimericholoenzyme results in increased degradation of the free subunitmonomers. Real-time PCR verified that there was no change inPPP2CA (C-subunit), PPP2R2A (B55a), or PPP2R5A (B56a)mRNA, confirming that the decreased protein resulted from aposttranslational mechanism (Fig. 4C). We then asked whetherthis protein loss could be reversed upon treatment with theproteasome inhibitor Velcade. After a 24-hour treatment, totalC-subunit protein was restored to a level comparable with that ofEGFP control lines (Fig. 4D and E), implicating proteasome-mediated degradation of C-subunit in P179R-expressing lines.Evaluation of a B-subunit isoform, B55a, revealed that its abun-dance was also increased with proteasome blockade in P179R-expressing cells, as well as in the EGFP and WT lines (Fig. 4D andE). This could suggest that at least some B-subunits are present inexcess and continuously turned over. To further verify decreasedsubunit stability in the presence of P179R-Aa, we investigatedprotein half-life using the translation inhibitor cycloheximide.P179R-Aa–expressing cells demonstrated an increased rate ofC-subunit degradation, represented by the slope of protein reduc-tion over time, which was significantly different from both EGFPandWT-Aa–transduced cells (Fig. 4F and G). Although EGFP and

WT demonstrated modest C-subunit degradation within theexperiment timeframe (�20%–30% reduced relative to time ¼0 hour), the higher rate of reduction in P179R allowed forcalculation of a half-life of 36 hours, at which point C-subunittotal protein was 50% reduced. The protein degradation rate ofB55a was also significantly increased in P179R-mutant cellsrelative to EGFP or WT cells (Fig. 4F and G). Finally, we evaluateda panel of PPP2R1A WT and P179R-mutant patient tumor speci-mens from the Case CCC tumor biobank for C- and B-subunitprotein levels; WT samples representative of both serous andendometrial carcinoma tumors were included (SupplementaryFig. S5A). We calculated the ratio of C:A (C-subunit to A-subunit)and B:A (B55a-subunit to A-subunit) to account for variation inthe basal level of A-subunit protein present in individual tumorspecimens. Overall, P179R-mutant tumors had lower levels of C-and B-subunit protein when compared with WT serous or endo-metrioid tumors (Supplementary Fig. S5A–S5C). The differenceswere statistically significant for B55a (Mut vs. Serous P¼ 0.0012;Mut vs. Endometrioid P ¼ 0.0028), and trended toward signif-icant for theC-subunit (Mut vs. Endometrioid P¼ 0.0688;Mut vs.Serous P ¼ 0.1838).

Given the PP2A Aa mutant–driven disruption of PP2A holo-enzyme assembly and reduction of catalytic subunit protein, andthat PP2A has prominent tumor-suppressive functions, we inves-tigated whether expression of P179R-Aa in the PPP2R1A WTUT89 cell line led to an alteration of in vitro or in vivo tumor cellgrowth. No significant difference in clonogenesis or tumorigen-esis was observed by colony formation assay or subcutaneousxenograft assay, respectively, when P179R-Aa was introducedinto a complete WT background (Supplementary Fig. S6A andS6B).

RestorationofWTPP2A function suppressesmalignant featuresof P179R-Aa–mutant cells

We derived another primary patient cell line, UT42, from apatient tumor that harbors the P179Rmutation endogenously, inorder to directly characterizemutant biology (Supplementary Fig.S2). This model system provides the environmental, cellular, andgenetic context native to a cell that acquired the P179R mutationas it underwent transformation. The UT42 cell line can be stablymaintained in cell culture for greater than 30 passages, and wasverified to retain the P179Rmutation by Sanger sequencing. UT42cells were transduced with V5-tagged WT-Aa or P179R-Aa toevaluate the impact of reconstitution with WT Aa protein ontumorigenic potential (Fig. 5A). Expression of WT-Aa proteinincreased C- and B-subunit protein, whereas expression of addi-tional P179R-Aa did not, suggesting that these subunits arestabilized when in the presence of the binding-competent WTprotein (Fig. 5B and C). In this model system, co-IP of the V5-tagged P179R-Aa displayed an 80% reduction in C-subunitbinding, which is consistent with data from UT89 (Fig. 5C andD). To assess the catalytic activity of reconstituted WT-Aa andcompare with that of mutant P179R-Aa protein, a phosphataseactivity assay was performed on the coimmunoprecipitants ofWT- or P179R-AaV5-tagged protein from theUT42 isogenic lines.Robust dephosphorylation of the substrate DiFMUP wasobserved for extracted WT-Aa protein bound to C- and B-subunits (Fig. 5E); this activity could be reversed by treatmentwith the serine/threonine phosphatase inhibitor OA (49). Bycontrast, significantly less dephosphorylation activity wasrecorded for extracted P179R-Aa and was comparable with that

Taylor et al.





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P179R-Aa expression induced a loss of PP2A C- and B-subunit stability. A,Western blots demonstrating loss of total C, B55a, and B56a protein upon expressionof the P179R-mutant Aa isoform that has impaired subunit binding. Triplicate lanes represent independent clonal lines generated from parental cells transducedin parallel wells. B,Quantification of total C-, B55a-, and B56a-subunits for selected clones used in subsequent experiments, presented as fold change relative toEGFP (n¼ 3). C,mRNA transcript levels of PPP2CA (Ca), PPP2R2A (B55a), and PPP2R5A (B56a) from real-time PCR, normalized to b-actin (n¼ 3). Statisticalsignificance for B and Cwas determined by the Student t test (WT or P179R vs. EGFP), with data graphed as mean� SD. D and E, RepresentativeWestern blotsand quantification of total C and B55a protein present following treatment with DMSO or Velcade (1.0 mmol/L) for 24 hours (hr; n¼ 3). Statistical significancewas determined by the Student t test (treated vs. untreated), with data graphed as mean� SD. F, RepresentativeWestern blots for change in total C and B55aprotein following treatment with cycloheximide (CHX; 100 mg/mL) for the indicated time. G, Linear regression analysis was performed on Ln-transformedWestern blot densitometries to represent the change in protein abundance across timepoints. Graphs represent the mean� SEM; tables provide the slope� SEof the best-fit line (n¼ 3). Details of the statistical analysis of cycloheximide data are provided in the Supplementary Methods. � , P� 0.05; �� , P� 0.01;�� , P� 0.001; and n.s., not significant.






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Restoration of C-subunit protein and PP2A catalytic activity upon expression ofWT-Aa in a P179Rþ tumor-derived cell line, UT42. A, Sanger sequencing of UT42cell lines transduced with expression vectors for EGFP, WT-Aa, or P179R-Aa. B, RepresentativeWestern blots demonstrate significantly increased C and B55aprotein with expression ofWT-Aa. Triplicate lanes are independent clonal lines generated from parental cells transduced in parallel wells. Graphed is the totalC-subunit protein for clones selected for subsequent experiments (n¼ 3). Data are presented as the mean� SD fold change relative to EGFP, and statisticalsignificance was determined by the Student t test (WT or P179R vs. EGFP). C,Western blots of co-IP isolates from the UT42 isogenic lines show that theexpressedWT-Aa protein is capable of binding to other PP2A C- and B-subunits; expressed P179R-Aa protein does not. D, Percent C-subunit binding of P179R-Aa relative toWT from co-IP in the UT42 cell lines (n¼ 3). Data presented as mean� SD, with significance determined by the Student t test. E, Co-IP isolates ofWT or P179R-Aa–containing complexes were assessed for phosphatase activity using a DiFMUP-based fluorescence assay (n¼ 3), whereinWT-Aa isolatesdisplayed robust dephosphorylation activity. This phosphatase activity could be blocked by OA treatment (OA, 50 nmol/L). Data were analyzed by two-wayANOVA (P < 0.0001; dF¼ 28) with Tukey post hoc t tests. F, Expression ofWT-Aa results in dephosphorylation of the PP2A substrates b-catenin and GSK3b.Images are representative ofWestern blotting results from three independent biological replicates (n¼ 3). G, Representative images and quantification ofcolony formation by UT42 cells expressing EGFP, WT-Aa, or P179R-Aa (n¼ 3). Data presented as mean� SD. Statistical significance was determined by theStudent t test (WT or P179R vs. EGFP). � , P� 0.05; �� , P� 0.01; �� , P� 0.001; �� , P� 0.0001; n.s., not significant.

Taylor et al.





seen with OA-mediated PP2A inhibition, suggesting a near-complete loss of catalytic activity with this recurrent, patient-derived Aa-mutant protein. Phosphatase activity has been nor-malized to the relative amount of C-subunit in the pull-downisolate, confirmed by Western blotting, to account for thedecreased catalytic C-subunit binding to P179R-Aa. Finally,expression of WT-Aa led to dephosphorylation of the establishedPP2A substrates b-catenin and GSK3b at the specific residuesagainst which the PP2A-B55a and PP2A-B56d holoenzymes,respectively, have previously demonstrated phosphatase activity(Fig. 5F; refs. 50–52). Consistent with the literature, dephosphor-ylation of b-catenin led to increased total protein as this eventprotects it from degradation.

A colony formation assay was used to assess the impact ofWT-Aa expression on in vitro clonogenic growth. UT42 cellsreconstituted with WT-Aa protein were significantly impaired intheir ability to form colonies, whereas expression of additionalmutant P179R-Aa protein did not affect clonogenesis comparedwith control (Fig. 5G).

Using both genetic and pharmacologic approaches, we soughtto determine the sensitivity of UT42 tumor growth to restorationof PP2A activity. First, UT42 cells that were reconstituted withcatalytically active,WT Aa protein were injected s.c. in the flank offemale mice. Tumor growth was compared with that of controlEGFP-expressing cells (Fig. 6A). Expression of WT-Aa proteinsignificantly inhibited subcutaneous tumor growth.

We next employed an orthotopic xenograft model withtumor cell injection into the uterus (Fig. 6B). In a pilot studywith parental UT42 cells, all animals developed primary tumorswithin the uterus by 8 weeks, as well as intraperitoneal met-astatic nodules; no lymph node enlargement was discerned.This model thus allows for assessment of both primary tumorgrowth within the native environment of the uterus corpus, aswell as cell metastatic potential. We carried out the full exper-iment under the same experimental conditions, injecting UT42EGFP or WT-Aa cells into the left uterine horn of female NSGmice. At study end, tumor burden was evaluated macroscop-ically through gross inspection of the isolated gynecologic tract,as well as inspection of the pelvic cavity, abdominal cavityorgans, and lungs for metastatic nodules. Tissue was collectedfor further microscopic evaluation and verification of tumorcell presence. At 8 weeks, 4 of 7 EGFP animals had tumor fillingthe entirety of the injected uterine horn, and the remaining 3animals had partial tumor presence visible macroscopically as amass along the uterine wall (Fig. 6B; Supplementary Fig. S6Cand S6D). By comparison, no WT animals had tumor filling theuterine cavity; four had partial tumor presence along theuterine wall, and the remaining three had no macroscopicallydiscernable tumor presence. Reduced tumor formation resultedin a significantly reduced uterus weight, despite apparenthydrometra (Fig. 6C). WT animals also demonstrated a signif-icant decrease in metastatic burden, as determined by metas-tasis nodule weight and nodule count (Fig. 6C and D). In bothgroups, metastatic nodules were predominantly peritoneal andsurrounded by subperitoneal fat. No organ-invasive metastaseswere identified in WT animals. By contrast, 2 EGFP animalsdisplayed metastatic nodules that were invasive into the liverparenchyma, and 1 animal had a retroperitoneal metastasisattached to the kidney capsule (Supplementary Fig. S6D andS6E). It should be noted that it is not possible to separate thedecreased occurrence of metastasis from the decreased primary

tumor burden of WT animals as an independent phenotype inthis study. We did however perform a scratch wound assay toassess whether the WT-Aa cells may be impaired in migratoryfunction and observed no significant difference from EGFP cells(Supplementary Fig. S6F).

In H&E-stained sections, tumor growth was found to expandwithin the endometrial layer of the uterus and through themyometrium, frequently present within parametrial soft tissueand occasionally encasing adnexal structures (Fig. 6E). Growth inthe outer uterine wall, subserosa, and parametrial soft tissue wasobserved in animals from both experimental groups. This growthpattern mirrors typical tumor growth observed in human tumorspecimens. One additional WT animal was found to have micro-scopic tumor foci that were not apparent during gross evaluation.The LLDwas calculated for each animal as an aggregatemeasure oftumor foci size. Expression ofWT-Aa led to amarked reduction intumor foci size relative to the tumors formed by EGFP-expressingcells (Fig. 6F).

Finally, we sought to evaluate the sensitivity of the UT42P179R mutant tumor to a pharmacologic method of PP2Areactivation. We have previously reported on a novel series ofSMAP that demonstrate in vivo suppression of tumor growth viaactivation of PP2A (19, 53, 54). SMAP-061 and SMAP-1154 aretwo lead compounds of the SMAP series, which was generatedthrough reverse engineering of tricyclic neuroleptics to removeanti-CNS toxicity and enhance antiproliferative properties(structures published in ref. 19; ref. 55). Through in vitro char-acterization, the UT42 cell line demonstrated robust growthinhibition with SMAP treatment and displayed an IC50 that wassignificantly lower than three other PPP2R1A WT endometrialcarcinoma lines, including UT89 (Supplementary Fig. S7A).Relative to UT89, the UT42 cell line also demonstrated greatersensitivity and marked growth impairment in the face of SMAPtreatment during a 2-week colony formation assay (Supple-mentary Fig. S7B).

To investigate SMAP response in vivo, PDXs of the UT42tumor were implanted subcutaneously in the flank of femalemice and treated with vehicle (DMA), 50 mg/kg SMAP-1154, or100mg/kg SMAP-1154 once daily. Tumor growth was markedlyreduced by SMAP treatment at both doses (Fig. 6G). Animalsreceiving SMAP treatment did not display signs of weight loss(Fig. 6H) or other side effects during monitoring. From theinitiation of treatment to study end, vehicle-treated tumorsdemonstrated robust increases in tumor volume, whereas min-imal change in tumor volume was observed with SMAP treat-ment (Fig. 6I). Critically, animals in the SMAP treatment groupsexhibited a greater rate of survival (Fig. 6J), as tumors receivingvehicle treatment grew rapidly to meet morbidity criteria foranimal euthanasia. Evaluation of protein isolates collectedfrom vehicle- or SMAP-treated tumors confirmed dephosphor-ylation of the PP2A substrates Akt, GSK3b, and c-Myc (Supple-mentary Fig. S7C and S7D). Alteration of these targets isconsistent with our previous works investigating the treatmentof tumors with SMAPs (19, 53, 54). Overall, the UT42 tumorharboring a P179R mutation was highly responsive to SMAPtreatment, which significantly reduced its growth in vivo.

DiscussionPP2A provides critical regulatory activity that counter-balances

kinase-mediated phosphorylation to maintain appropriate cell






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growth, regulate cell division, and prevent tumor development. Arecurrent mutation of the PP2A Aa scaffolding subunit(PPP2R1A) is present in approximately 30% of high-grade sub-types of endometrial carcinoma, with mutation at the P179 siteaccounting for approximately 20% to 25%. The UT42 patient-derived cell model harbors an endogenous P179R mutation andthus provides a unique system in which to investigate mutantbiology as this tumor developed in the immediate context ofsomatic PPP2R1A mutation. The striking impairment of tumorformation and metastasis that occurred when WT Aa protein wasrestored provides evidence that functional disruption of PP2A iscritical to this tumor and its malignant features. In a similarmanner, tumor growth was significantly reduced when treatedwith a pharmacologic PP2A activator, again highlighting sensi-tivity of the P179Rmutant UT42 tumor to PP2A activity. Overall,PP2A's robust roles as a suppressor of transformation and tumordevelopment have been well-documented and reviewed in theliterature, to which our outcomes add further emphasis on itsimportance as a tumor suppressor in high-grade endometrialcarcinoma (56–58).

P179 site mutations are notable for displaying marked diseasespecificity, occurring almost exclusively in endometrial carcinomaand the serous carcinoma and carcinosarcoma subtypes. Thispattern suggests a role for PP2A as a disease driver, in which thebiochemical consequence of mutating this residue is specificallyadvantageous to endometrial tumorigenesis. Consistent with thisperspective, a large-scale sequencing study has shown thatPPP2R1Amutations are somatic and truncal, and so are presumedto be acquired during the early processes of malignant celltransformation (9). Several research groups have investigatedPP2A as a tumor suppressor whose functional loss contributesto cell transformation. Interestingly, a majority of developedmodels rely on coinactivation of p53 with PP2A for completetransformation; meanwhile, TP53 and PPP2R1A P179 mutationshighly co-occur and are both truncal in these otherwise lowmutational burden endometrial carcinoma tumors (8–10, 59).P179 mutation may therefore be a mechanism through whichmodels of PP2A in transformation bear out in endometrialcarcinoma.

To understand how the P179Rmutant alters PP2A function, webegan with investigation of PP2A subunit binding. Our findingsare in agreement with previous literature that identified disruptedPP2A subunit interactions due to P179R PPP2R1A muta-tion (17, 18). The consequence for unbound B- and C-subunits,an increased rate of protein degradation, is a novel finding for thiscancer-associated mutation but is in support of a standing viewthat monomeric PP2A B- and C-subunits are less stable than theirtrimeric counterparts (11, 60). Together, these findings suggestthat acquisition of a P179R mutation will impede assembly of

holoenzymes containing catalytic and regulatory subunits andthereby induce a corresponding loss of canonical PP2A functionwithin the cell.

The P179 residue resides at the A/B subunit interface, andalteration of its side chain chemistry could reasonably bepredicted to directly impair B-subunit binding. On the otherhand, significant loss of C-subunit binding suggested to us thatthis mutation had a larger, global effect on the scaffoldingprotein's conformation. By utilizing crystallography and molec-ular dynamics modeling, we provide context to the interactomedataset by detailing the structural alterations induced by theP179R substitution. Simulations of the WT protein revealproline residue 179 as a site of cis-trans isomerization that canconfer dynamic flexibility to the A-subunit scaffolding protein.The substituted arginine changed the dominant isomerizationstate and introduced new interresidue interactions within theprotein tertiary structure. The P179R substitution modifies theA-subunit's most stable, and preferentially adapted, conforma-tion in a manner that is unfavorable to C-subunit binding, asevidenced by Aa/C complex simulations for the mutant iso-form as well as its increased binding energy requirement. Inaddition, we were able to resolve the crystal structure of themutant protein at a 3.4 Å resolution. Although publishedcrystallographic structures have resolutions ranging from 1 to4 Å, with an average resolution of approximately 2.2 Å, manystructures, especially those with all-helical secondary structuressuch as the PP2A Aa protein, can be clearly resolved in 3 to 4 Åwithout ambiguity. This is because alpha-helices have morecharacteristic electron densities and have well-defined main-chain structural restrictions. P179R-Aa is the first resolvedcrystal structure for a cancer-derived mutant PP2A protein, andit underscores that mutation is a pathogenic mechanismthrough which cancer cells can disrupt the PP2A structure.

Importantly, our work suggests that a previously describedmechanism of PP2A inactivation by PPP2R1A hotspot mutationmay not hold true for P179R (18). Although some PPP2R1Amutations display increased binding to the PP2A inhibitor TIPRLandmaybe inactivated throughadominant-negativemechanism,P179R did not display an increase in TIPRL binding in our modelsystems. This included protein co-IP in both a WT serous endo-metrial carcinoma cell line (Fig. 2A), as well as within a cell linethat itself harbors the P179Rmutation endogenously (Fig. 5C). Itis possible that the conflicting observations in P179R interactomereflect on the context specificity of this mutation; however, atargeted investigation would be needed to lend more conclusiveclarity to this discrepancy.

There is a strong clinical need for new therapeutic options inendometrial carcinoma, and in particular for high-grade sub-types like serous endometrial carcinoma that portend a poor

Figure 6.PP2A activation in UT42 suppressed tumorigenesis andmetastasis in vivo.A, Tumor growth following s.c. injection of EGFP- orWT-Aa–expressing UT42 cellsinto the animal's hind flank (n¼ 11). B, Schematic of the intrauterine cell injection method (adapted from ref. 21) with representative images of primary tumorformation in the extracted gynecologic tract (images for all animals presented in Supplementary Fig. S6C). C,Weight of the injected left uterine horn, and ofcollected metastatic nodules, isolated frommice at 8 weeks after injection (n¼ 7).D, Number of metastatic nodules per animal and group. E, H&E-stainedsections of representative EGFP andWT primary uterine tumors at lowmagnification (top; scale bar, 1 mm) and high magnification (bottom; scale bar,100 mm). F, The LLDwas calculated as the sum of the lengths of all tumor foci observed within the sampled H&E-stained tissue section. G, Tumor growth ofsubcutaneous UT42 PDX implants treated with vehicle (DMA; n¼ 8), 50 mg/kg SMAP (n¼ 6), or 100 mg/kg SMAP (n¼ 6). H, Animal weights recorded acrossthe duration of treatment. I,Waterfall plot presenting the percent change in tumor volume between the start of treatment and treatment day 15. J, Kaplan–Meiercurves for animal survival within each treatment group. Data for all in vivo experiments are presented as the mean� SEM, with statistical significancedetermined by the Student t test, except for survival curves that were evaluated for significance by the log-rank (Mantel–Cox) test. � , P� 0.05; �� , P� 0.01;�� , P� 0.001; n.s., not significant.






prognosis due to tumor recurrence. Recently, immunotherapyhas become an exciting prospective therapeutic opportunity formicrosatellite instable endometrial carcinoma tumors due totheir abundance of neoantigens (61). However, tumors thatharbor P179 mutations are unlikely to be amenable to theimmunotherapy approach as we show this mutation occursexclusively in low mutational burden, MSI-negative tumortypes. The outcomes of the in vivowork presented here highlighta path forward for use of a targeted therapeutic approach thatcapitalizes on PP2A reactivation.

Finally, in addition to cancer, instances of germline PPP2R1Amutation have been reported in association with developmentaland intellectual disabilities (62, 63). AP179Lmutationwasoneofseveral missense mutations identified. Whether this confersincreased risk for cancer is unknown. From our results, one couldreasonably predict these mutations lead to significant disruptionof PP2A physiologic activity within neuronal cells.

Overall, this body of work suggests a loss of function ofPP2A tumor-suppressive activity due to P179R mutation ofthe Aa-subunit. "Rescue" through expression of holoenzyme-forming and catalytically competent WT Aa protein, or throughpharmacologic PP2A activation, suppressed tumorigenesis.These findings support PPP2R1A P179R mutation as key driverof disease in the 20% to 30% of high-grade endometrialcarcinoma tumors that harbor one, and present pharmacologictargeting of PP2A as a potential therapeutic direction for thispatient population.

Disclosure of Potential Conflicts of InterestK. Resnick reports receiving honoraria from the speakers' bureau of Clovis.

G. Narla is CSO at RAPPTA Therapeutics, has an ownership interest (includingstock, patents, etc.) in RAPPTA Therapeutics, and is a consultant/advisory boardmember for HERA. The Icahn School of Medicine at Mount Sinai has filedpatents covering composition ofmatter on the smallmolecules disclosed hereinfor the treatment of human cancer andother diseases (International ApplicationNumbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358B2). Mount Sinai is actively seeking commercial partners for the furtherdevelopment of the technology. G. Narla has a financial interest in the com-mercialization of the technology. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: S.E. Taylor, C.M. O'Connor, S. Haider, A. DiFeo,G. NarlaDevelopment of methodology: S.E. Taylor, G. Shen, D. Leonard, W. Xu,S. Haider, A. DiFeo, G. NarlaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.E. Taylor, C.M. O'Connor, Z. Wang, H. Song,D. Leonard, J. Sangodkar, C. LaVasseur, S. Avril, S. Waggoner, K. Zanotti,A.J. Armstrong, C. Nagel, K. Resnick, W. Xu, A. DiFeoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.E. Taylor, Z. Wang, H. Song, D. Leonard,J. Sangodkar, S. Avril, W. Xu, S. Haider, A. DiFeo, G. NarlaWriting, review, and/or revision of the manuscript: S.E. Taylor,C.M. O'Connor, Z. Wang, D. Leonard, J. Sangodkar, C. LaVasseur, S. Avril,W. Xu, S. Haider, G. NarlaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.E. Taylor, H. Song, W. Xu, S. HaiderStudy supervision: S.E. Taylor, M.W. Jackson, W. Xu, S. Haider, G. Narla

AcknowledgmentsThe authors would like to thank the Case CCC, which supports the Gyne-

cologic Tumor Biobank, for the invaluable resources provided by this work;Kristen Weber Bonk and the CWRU Athymic Animal and Xenograft Core fortheir continued support with the in vivo studies; Daniela Schlatzer and theCWRU Proteomics Core for support with proteomics work; and finally, RichardLee and the CWRU Light Microscopy Imaging Core. The Imaging Core's LeicaSCN400 slide scanner utilized for this work is made available through the NIHOffice of Research Infrastructure (NIH-ORIP) Shared Instrumentation Grant(S10 RR031845). Funding for this work was provided by grants from theNIH-NCI to G. Narla (R01 CA181654), A. DiFeo (R01 CA197780), and S.E.Taylor (F30 CA224979); the Department of Defense to A. DiFeo (OC150553);and The Young Scientist Foundation to A. DiFeo. S.E. Taylor andD. Leonard areadditionally supported by T32 GM007250 (NIH-NIGMS), and C.M. O'Connorby T32 GM008803 (NIH-NIGMS). S. Avril is supported by a Clinical andTranslational Science Award KL2 TR0002547 (NIH-NCATS).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 17, 2019; revised April 12, 2019; accepted May 22, 2019;published first May 29, 2019.

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2019;79:4242-4257. Published OnlineFirst May 29, 2019.Cancer Res Sarah E. Taylor, Caitlin M. O'Connor, Zhizhi Wang, et al. Endometrial TumorigenesisProtein Structure and Impairs PP2A Enzyme Function to Promote

-Subunit Mutation P179R AltersαThe Highly Recurrent PP2A A

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