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Translational Science

Small-MoleculeActivators of Protein Phosphatase2A for the Treatment of Castration-ResistantProstate CancerKimberly McClinch1, Rita A. Avelar2, David Callejas2, Sudeh Izadmehr1,5,Danica Wiredja4, Abbey Perl2, Jaya Sangodkar5, David B. Kastrinsky3,Daniela Schlatzer4, Maxwell Cooper2, Janna Kiselar4, Agnes Stachnik5, Shen Yao6,Divya Hoon5, Daniel McQuaid5, Nilesh Zaware3, Yixuan Gong1, David L. Brautigan7,Stephen R. Plymate8, Cynthia C.T. Sprenger8,William K. Oh1, Alice C. Levine6,Alexander Kirschenbaum9, John P. Sfakianos9, Rosalie Sears10, Analisa DiFeo11,Yiannis Ioannou5, Michael Ohlmeyer3, Goutham Narla2,11, and Matthew D. Galsky1

Abstract

Primary prostate cancer is generally treatable by androgen dep-rivation therapy, however, later recurrences of castrate-resistantprostate cancer (CRPC) that aremore difficult to treat nearly alwaysoccur due to aberrant reactivation of the androgen receptor (AR). Inthis study, we report that CRPC cells are particularly sensitive to thegrowth-inhibitory effects of reengineered tricyclic sulfonamides, aclass of molecules that activate the protein phosphatase PP2A,which inhibits multiple oncogenic signaling pathways. Treatmentof CRPC cells with small-molecule activators of PP2A (SMAP) invitro decreased cellular viability and clonogenicity and inducedapoptosis. SMAP treatment also induced an array of significantchanges in the phosphoproteome, includingmost notably dephos-

phorylation of full-length and truncated isoforms of the AR anddownregulation of its regulatory kinases in a dose-dependent andtime-dependent manner. In murine xenograft models of humanCRPC, the potent compound SMAP-2 exhibited efficacy compara-ble with enzalutamide in inhibiting tumor formation. Overall, ourresultsprovideapreclinicalproofof concept for theefficacyofSMAPin AR degradation and CRPC treatment.

Significance: A novel class of small-molecule activators of thetumor suppressor PP2A, a serine/threonine phosphatase that inhi-bitsmany oncogenic signaling pathways, is shown to deregulate thephosphoproteome and to destabilize the androgen receptor inadvanced prostate cancer. Cancer Res; 78(8); 2065–80. �2018 AACR.

IntroductionDepletion of circulating androgens is the standard first-line

treatment for metastatic prostate cancer and results in tumorregression and symptomatic improvement in the majority ofpatients. However, metastatic prostate cancer inevitably pro-gresses despite castrate levels of serum testosterone, a diseasestate known as castration-resistant prostate cancer (CRPC). Stud-ies in model systems, and in patients, have confirmed that theandrogen receptor (AR) signaling axis remains a key therapeutictarget in CRPC, and novel approaches to androgen biosynthesisinhibition and androgen receptor blockade have resulted inimproved patient outcomes (1, 2). Still, such treatments are notcurative and prostate cancers ultimately develop resistance via avariety of mechanisms including ligand-independent AR signal-ing or alternative pathway activation, highlighting the need fornew therapeutic approaches (3).

Protein phosphatase 2A (PP2A), a serine/threonine phospha-tase and bona fide tumor suppressor, has been implicated in thepathogenesis of CRPC. PP2A dephosphorylates a large number ofcritical oncogenic proteins including AKT1, ERK,MYC, BCL2, andothers (4). Indeed, PP2A has been shown to directly bind anddephosphorylate the AR (5). PP2A is frequently decreased orfunctionally inactivated in CRPC and decreased expression inhuman tumor specimens has been associated with inferior out-comes and enzalutamide resistance (6–8). Nonpharmaceuticalactivators of PP2A have demonstrated anticancer effects in

1Department of Medicine, Division of Hematology and Medical Oncology, TischCancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York.2Department of Medicine, Institute for Transformative Molecular Medicine, CaseWestern Reserve University, Cleveland, Ohio. 3Department of PharmacologicalSciences, Icahn School of Medicine at Mount Sinai, New York, New York. 4Centerfor Proteomics and Bioinformatics, CaseWestern Reserve University, Cleveland,Ohio. 5Department of Genetics and Genomic Sciences, Icahn School of Medicineat Mount Sinai, New York, New York. 6Department of Medicine, Division ofEndocrine, Diabetes andBoneDiseases, Icahn School ofMedicine atMount Sinai,New York, New York. 7Center for Cell Signaling, University of Virginia School ofMedicine, Charlottesville, Virginia. 8Department of Medicine, University ofWashington School of Medicine, Seattle, Washington. 9Department of Urology,Icahn School of Medicine at Mount Sinai, New York, New York. 10Department ofMolecular and Medical Genetics, Oregon Health and Science University, Port-land, Oregon. 11Case Comprehensive Cancer Center, Case Western ReserveUniversity, Cleveland, Ohio.

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

K.McClinch,R.A.Avelar,D.Callejas, andS. Izadmehrcontributedequally to thisarticle.

Corresponding Authors: Matthew D. Galsky, Department of Medicine, Divisionof Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School ofMedicine at Mount Sinai, 1 Gustave Levy Place, New York, NY 10029. Phone: 212-659-5426; Fax: 212-659-5533; E-mail: [email protected]; GouthamNarla, Case Comprehensive Cancer Center, Case Western Reserve University,2103 Cornell Road, Cleveland, OH 44106. Phone: 216-368-3111; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-0123

�2018 American Association for Cancer Research.

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prostate cancer model systems (9–15). Together, these findingspoint to therapeutic activation of PP2A as a novel strategy for thetreatment of prostate cancer.

Tricyclic neuroleptics have been reported to activate PP2A incells through direct binding of the PP2A Aa subunit, thoughclinical development of such molecules for cancer therapy islimited by central nervous system toxicity (16, 17). We havedeveloped first-in-class small-molecule activators of PP2A(SMAP) by repurposing and reengineering FDA-approved tri-cyclic neuroleptics (18). By replacing the basic amine with aneutral polar functional group, the central nervous systemeffects were abrogated and further chemical derivatization hasimproved anticancer potency (18). Through binding studiesusing a tritiated version of SMAPs, in silico docking calculations,photo-affinity labeling experiments, and hydroxyl radical foot-printing studies, we have shown that these SMAPs directly bindto the PP2A Aa subunit (19). The postulated mechanism ofaction is that SMAP binding promotes allosteric conformation-al changes, leading to the activation of the phosphatase anddephosphorylation of key substrates (19). Given the criticalrole of the AR, a known PP2A substrate in CRPC, as well as therole of PP2A in the pathogenesis of prostate cancer, we exploredthe effects of SMAPs in cell culture and in vivo model systemsof CRPC.

Materials and MethodsCompound synthesis

All compounds were synthesized in the laboratory of Dr.Michael Ohlmeyer at the Icahn School of Medicine at MountSinai (New York, NY). Compounds were stored at roomtemperature.

Cell cultureLNCaP (catalog no. CRL-1740, lot no. 59453491) and 22Rv1

(catalog no. CRL-2505, lot no. 60437301) cell lines were pur-chased from the ATCC and authenticated by ATCC (STR DNAprofiling). The LNCaP/AR cell line was a generous gift fromDr. Charles Sawyers (Memorial Sloan Kettering Cancer Center,New York, NY) and was authenticated by ATCC STR DNAProfiling Authentication Services (ATCC) with the FTA SampleCollection Kit for Human Cells (catalog no. 135-XV3). All celllines included in the Oncopanel 240 Anti-Cancer Drug ProfilingAssay (Eurofins Panlabs, Inc.) were authenticated throughSTR DNA profiling by Genetica DNA Laboratories. Mycoplasmatesting was performed routinely with Lonza MycoAlert Myco-plasma Detection Kit as per the manufacturer's protocol (catalogno. NC9922140, Thermo Fisher Scientific). LNCaP, 22Rv1, andLNCaP/AR cells were cultured in RPMI1640 medium (ThermoFisher Scientific) with 10% fetal bovine serum (HyClone)and 0.5% penicillin–streptomycin (Thermo Fisher Scientific).All cell lines were maintained at less than 80% confluence and25 passages. Cells were maintained at 37�C with 5% CO2.SMAPs (dissolved in DMSO) were diluted to a stock concen-tration of 80 mmol/L. Dilutions to the needed concentrationswere made in RPMI1640 (Thermo Fisher Scientific).

MTT and colony formation assaysCells were treated with SMAPs and screened for cell viability

through the MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich). For clonogenic

assays, cells were plated at a low density in 6-well plates. After 48hours, cells were treated with DMSO (Simga-Aldrich)or increasing concentrations of SMAPs for 10–12 days. Drugmedium was refreshed every 48 hours. Cells were fixed andstained with 1% crystal violet solution (Sigma-Aldrich). Quanti-fication was performed through the cell counter function onImageJ (imagej.nih.gov/ij/).

Western blottingCell protein was isolated with RIPA Lysis and Extraction Buffer

(Thermo Fisher Scientific). Isolated protein was quantified, nor-malized via Bio-Rad assay (Bio-Rad), run on a 12% SDS-PAGE(Invitrogen, Life Technologies), and transferred onto nitrocellu-lose membranes (Bio-Rad). The membrane was blocked with 5%nonfat milk (LabScientific Inc.) in Tris-buffered saline–Tween20 buffer. Antibody information is provided in SupplementaryMaterials and Methods.

Annexin stainingAnnexin V staining was performed using Annexin V conjugate

Alexa Fluor-488 (Invitrogen, Life Technologies) or 7-Aminoacti-nomycin D (7-AAD; Thermo Fisher Scientific) and Annexin bind-ing buffer (catalog no. V13246, Invitrogen, Life Technologies),according to the manufacturer's protocol. Cells were also stainedwith propidium iodide (Roche) to ascertain the DNA content andas a marker of cell death. Each experiment was performed intriplicate.

PhosphoproteomicsQuantitative global phosphorylation studies were performed

with LNCaP cells using an unfractionated label-free LC/MS-MSworkflow. Cells were treated with DMSO or 30 mmol/L SMAP for6 hours. After incubation, cells were harvested, pelleted, andwashed with PBS. Samples were lysed with 2% SDS solutioncontaining both protease (catalog no. P8465, Sigma Aldrich) andphosphatase inhibitors (PhosphoSTOP, Roche). All sampleswerequantified by the Bicinchoninic acid assay (BCA assay) andnormalized on the basis of total protein concentration beforeprocessing for global phosphoproteomics.Detergent removalwasperformed on 200 mL of the cell lysate using the FASP cleaningprocedure (20). Eight-hundred micrograms of each sample wasdigested using by a 2-step Lys-C/trypsin proteolytic cleavage. Eachdigest was equally split into two 400-mg samples to provide twotechnical replicates. Each technical replicate was subsequentlyenriched for phospho-peptides using commercially availableTiO2 enrichment spin tips (Thermo Fisher Scientific). The entiretyof the samples was phospho-enriched, with no residual amountsaved for parallel unenriched LC/MS-MS. A 3-hour LC/MS-MSmethod was performed using a UPLC system (NanoAcquity,Waters) that was interfaced to Orbitrap ProVelos Elite MS system(Thermo Fisher Scientific) to perform the LC/MS-MS data collec-tion. Clustering of all the peptide precursor ions across all thechromatographic analyses was performed using the peak align-ment algorithm of the Rosetta Elucidator software. Automateddifferential quantification of phospho-peptides was accom-plished using downstream quantitative analysis modules ofElucidator. MS/MS peak lists were generated and subsequentlysearched by Mascot version 2.4.0 (Matrix Science). The databaseused was human UniProt (538,585 sequences). Search settingswere as follows: trypsin enzyme specificity;mass accuracywindowfor precursor ion, 8 ppm; mass accuracy window for fragment

McClinch et al.

Cancer Res; 78(8) April 15, 2018 Cancer Research2066




ions, 0.6 Daltons; variable modifications including carbamido-methlylation of cysteines, phosphorylation of serine, threonine,and tyrosine and 1 missed cleavage. Peptide and protein identi-fications were integrated from the protein database search engineoutput with these quantifications. Fold changes were calculatedfrom themean peptide intensity of SMAP over themean intensityof DMSO. Statistical significance of abundance changes wasdetermined using Welch t test.

Bioinformatics analysesNetwork analysis. Data preprocessing was performed using R.An unfiltered list of phosphoproteins identified from phos-phoproteomics was searched against the HPRD binary pro-tein–protein interaction database, Release 9. Hits and theirinteractions were compiled into a .sif file and imported intoCytoscape 3.0.2 for downstream processing and image gener-ation. For proteins with multiple phosphosites detected, theresidue with the lowest P value was selected for P value andfold change illustrations. The MCODE plugin was used toextract clusters using the default settings except for a NodeScore Cutoff ¼ 0.5. Node degree was calculated using theNetworkAnalyzer tool.

Kinase-Substrate Enrichment Analysis. Calculations and plottingwere performed using R. Please refer to the original publication(21) for details on the formulas. The authors reported threevariations on the algorithm; our analysis was based on the thirdmethod described in their Materials and Methods. We assignedkinase–substrate links based on the Kinase SubstrateDataset fromPhosphoSitePlus (October 2015 release), search restrictedto human proteins. The calculated P values were adjusted formultiple hypotheses testing using the p.adjust function in R(method¼"fdr") and reported in the Supplementary Table S1.

Establishment of tumor xenografts and in vivo treatmentstudies

Studies were conducted after Institutional Animal Care andUse Committee approval at the Icahn School of Medicine atMount Sinai [protocol: small-molecule activators of PP2A(SMAP) for prostate cancer therapy, LA13-00005]. Animal useand care was in strict compliance with institutional regulatorystandards and guidelines. Eight-week-old male SCID/NCr(BALB/c background) noncastrated and castrated mice (strainno. 01S11, NCI-Frederick Mouse Repository, Frederick, MD)were used for treatment studies. Studies were nonblinded.MDV3100 (enzalutamide, catalog no. S1250) was purchasedfrom Selleck Chemicals. LNCaP/AR cells (5 � 106) weresuspended in 200 mL of 1:1 RPMI1640/Matrigel (BD Bio-sciences) and were injected subcutaneously into the right flankof the mice. When tumor volumes reached an average of 100–250 mm3 mice were randomized to treatment groups. Tumorvolumes and body weights were measured every other daythroughout the study. Tumor volume was assessed by a digitalcaliper and determined using the formula: length � width2/2.Percentage of mice body weights during treatment were cal-culated as: weight at each time-point/initial weight � 100.Mice were treated by oral gavage with vehicle control,MDV3100 (100 mg/kg once daily), SMAP (100 mg/kg or400 mg/kg twice daily), or SMAP-2 (30 mg/kg or 100 mg/kgtwice daily). MDV3100, SMAP, and SMAP-2 for efficacy studieswere formulated in a homogenous suspension with 0.1%

Tween-80 (lot no. MKBP0682V, Sigma-Aldrich)/0.5% NaCMC(lot no. SLBF3845V, Sigma-Aldrich) in diH2O. SMAP-2 for thepharmacodynamic study was prepared in a N,N-Dimethylace-tamide (DMA)/Kolliphor HS-15 (Solutol)/diH20 solution.Animals were observed for signs of toxicity (i.e., mucousdiarrhea, abdominal stiffness, and weight loss). Blood andtumor tissue was harvested 2 hours post-final dose of treat-ment. Tumors were formalin-fixed for IHC or snap frozen inliquid nitrogen for immunoblotting. Serum was submitted fortoxicology testing at IDEXX Laboratories.

Histology and IHCIsolated tissue was fixed in 10% buffered formalin phosphate

(Thermo Fisher Scientific, catalog no. SF100-4), transferredto 70% ethanol, and blocked in paraffin. Serial tissue sections(5-mm thickness) were cut from the paraffin-embedded blocksand placed on charged glass slides. Tumor sections were stainedwith hematoxylin and eosin (H&E) and PCNA (Abcam, catalogno. ab92729). Sections were deparaffinized with xylene, rehy-drated through graded alcohol washes, and followed byantigen retrieval in a pressure cooker (Dako/Agilent Techno-logies) in citrate buffer (10 mmol/L, pH 6.0, Vector Laborato-ries). Slides were incubated in hydrogen peroxide–methanoland incubated in normal goat serum/PBS. Primary antibodywas applied overnight at 4�C. DAB substrate was appliedfollowed by counterstaining in hematoxylin. The ApopTagFluorescein In Situ Apoptosis Detection Kit (TUNEL; catalogno. S7110, Millipore-Sigma) was used according to the man-ufacturer's protocol. Vectashield Mounting Medium with pro-pidium iodide (catalog no. H-1300), Vector Laboratories) wasused for counterstaining. Bright-field and fluorescent imageswere captured using a Zeiss Axioplan 2IE microscope. Imagingwas performed at the Icahn School of Medicine at Mount SinaiMicroscopy CORE. ImageJ software with cell counter functionwas used to quantify stained cells (imagej.nih.gov/ij.).

Quantitative real-time quantitative PCRTotal RNA was extracted using the QiaShredder Kit (Qiagen)

and the RNeasy Kit (Qiagen). cDNA synthesis was carried outusing the iScript cDNA Synthesis Kit (Bio-Rad) as per the man-ufacturer's instructions. Sequences can be found in Supplemen-tary Materials and Methods. Real-time PCR was performed withSYBR green PCRMaster Mix (Applied Biosystems) on the AppliedBiosystems 7900HT Fast Real-Time PCR System.

Statistical analysesGraphPad Prism 6 or 7 software (GraphPad Software Inc.)

were used for statistical analysis. The results are presented asa mean � SD. In vitro experiments were performed in triplicatefor three biological replicates. One-way ANOVA was utilized tocompare mean values and Tukey or Dunnett test were appliedwhere appropriate. P values � 0.05 were considered statisti-cally significant.

ResultsProstate cancer cell lines are sensitive to SMAPs

To investigate the broad utility of our reengineered tricyclicstoward various cancer cell lines, an Oncopanel 240 anticancerdrug-profiling screen was performed by Eurofins Pathlab, Inc.The tool compound, TRC-382, displayed growth-inhibitory

PP2A Activation for the Treatment of Prostate Cancer

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properties in several tumor types, including prostate cancer(Fig. 1A; Supplementary Fig. S1). Although TRC-382 exhibitedbroad activity across a variety of cancer types, which is perhapsnot surprising given the range of oncogenic proteins that serveas PP2A substrates, prostate cancer cell lines were among themost sensitive. Furthermore, among the prostate cancer celllines, the AR-expressing cell lines, 22Rv1 and LNCaP, exhibitedthe greatest sensitivity to TRC-382 (Fig. 1A), whereas growthwas not inhibited in the immortalized but nontumorigenic cellline, BPH-1. On the basis of these findings and prior researchindicating that nonpharmaceutical activators of PP2A haveanti-prostate cancer activity (9–15) and literature indicatingthat several PP2A substrates are implicated in the pathogenesisof prostate cancer (7, 22–25), further functional studieswere pursued in prostate cancer. Subsequent studies focusedon AR-expressing prostate cancer and were performed withSMAP and SMAP-2, structurally similar variants of TRC-382containing a hydroxylated linker that confers improved bio-availability (Supplementary Fig. S2; ref. 18). LNCaP and 22Rv1cells were treated with increasing concentrations of SMAP for48 hours. SMAP treatment decreased viability in both cell lineswith IC50s of 16.9 mmol/L (LNCaP) and 14.1 mmol/L(22Rv1; Fig. 1B). As a pharmacologic control, these cells werealso treated with increasing doses of TRC-766, which is struc-turally similar to SMAP but biologically inactive. TRC-766 lacksa key N-H sulfonamide hydrogen bond donor function likelynecessary for the interaction with PP2A's catalytic subunit(Supplementary Fig. S2). While TRC-766 still binds PP2A, itdoes not activate the phosphatase and therefore it was postu-lated to not affect cell viability. Consistent with this hypothesis,biologically inactive TRC-766 displayed negligible effects oncell viability (Fig. 1B). Next, clonogenic assays were performedon LNCaP and 22Rv1 cell lines. Cells plated at low density weretreated with increasing doses of SMAP or TRC-766 for 12 days.Consistent with its effects on viability, SMAP significantlyinhibited cell survival, which was dose-dependent, whereasTRC-766 had minimal to no effect (Fig. 1C; SupplementaryTables S2 and S3). Subsequently, Annexin V staining of LNCaPand 22Rv1 cells was performed with increasing doses of SMAPfor 24 hours. SMAP induced an increase in Annexin V positivityin LNCaP and a statistically significant increase in 22Rv1 cells,suggesting that treatment with increasing doses of SMAP caninduce cell death (Fig. 1D; Supplementary Table S4). Theseresults were confirmed by probing for cleaved PARP and cas-pase-3 cleavage, which were detected by Western blot analysisin both LNCaP and 22Rv1 cells at a dose of 30 mmol/L of SMAPas early as 6 hours posttreatment (Fig. 1E).

Phosphoproteomic analysis reveals AR perturbation uponSMAP treatment

PP2A dephosphorylates and inactivates numerous sub-strates. To define perturbations in phosphorylation resultingfrom SMAP treatment, an analysis of the global phosphopro-teome was performed. LNCaP cells were treated with 30mmol/L of SMAP for 6 hours versus control. Extracted proteinswere digested and phospho-enriched prior to LC/MS-MSanalysis under the label-free protocol. A total of 3,051 uniquephosphosites (p-sites), which mapped to 2,735 phosphopep-tides and 1,496 phosphoproteins, were identified. Overall,927 p-sites (mapping to 651 phosphoproteins) met a P < 0.05Welch t test criterion and thus were deemed significantlyperturbed by drug treatment as compared with control. Fromthis list, 673 p-sites were downregulated upon treatment, and254 were upregulated (Fig. 2A). This count suggests thatSMAP treatment predominantly led to a decrease in phos-phorylated peptides.

From this dataset, we then applied two bioinformatics toolsto extract biological patterns in a relatively unbiased manner.First, we utilized a network model to identify candidate masterregulators that are altered by SMAP treatment. We overlaid allour identified phosphoproteins onto a protein–protein inter-action (PPI) network built from relationships documented inthe Human Protein Reference Database, Release 9 (26). Theresulting PPI contained 613 unique phosphoproteins linkedby 982 interactions. We then reduced this large PPI into a moreconcise subnetwork using MCODE, a tool designed to enrichfor tightly interconnecting proteins that are functionally relat-ed (27). We then profiled members of the top-scoring clusterto find those with the highest number of interactions (degree).These so-called "hub proteins" are often essential nodes ofregulation within the cell (27). Interestingly, AR had thehighest degree within our subnetwork (Fig. 2B) and acrossthe original network (Supplementary Tables S5 and S19).Given this finding and the observation that this protein wassignificantly downregulated upon SMAP treatment, wehypothesized that our compound's activity disrupts AR-relatedprocesses.

In addition to the network analysis, we also sought to profiledifferential signaling fluxes in response to SMAP treatment.Kinase-Substrate Enrichment Analysis (KSEA) scores each kinase'srelative activity output based on the collective phosphorylationfold change of its identified substrates (28). A negative valueindicates that themajority of the kinase's substrates had decreasedphosphorylation upon treatment, thereby suggesting that itsoverall signaling output is decreased compared with control. This

Figure 1.Prostate cancer cell lines are sensitive to SMAPs. A, Prostate cancer cell lines' IC50s of TRC-382 as determined by Oncopanel 240 (Eurofins Pathlab, Inc.) screenof 240 cancer cell lines. IC50s are shown relative to the average IC50 (19.6 mmol/L) for all 240 cell lines treated with TRC-382. B, LNCaP and 22Rv1cells were treated with vehicle, 10, 20, 30, and 40 mmol/L of SMAP or TRC-766, a biologically inactive analogue of SMAP, and cell viability was measuredat 48 hours by MTT analysis. C, Clonogenic assay of LNCaP and 22Rv1 cells treated with vehicle control, 5, 7.5, 10, 12.5, and 15 mmol/L of SMAP (top) or TRC-766(bottom) for 2 weeks. Quantification of colony formation is shown. Data are means � SD. ANOVA performed for LNCaP cells treated with SMAP (P < 0.0001),LNCaP cells treated with TRC-766 (P ¼ 0.9993), 22Rv1 cells treated with SMAP (P < 0.0001), and 22Rv1 cells treated with TRC-766 (P ¼ 0.9993). Full details ofANOVA and Dunnett test are provided in Supplementary Tables S2 and S3. D, Annexin V/7AAD staining of LNCaP cells and 22Rv1 cells after 24 hoursof exposure to vehicle control, 10, 20, and 30 mmol/L of SMAP. Quantification of Annexin V–stained cells is shown. Data are means � SD. ANOVA performedfor LNCaP cells treated with SMAP (P ¼ 0.1015) and 22Rv1 cells treated with SMAP (P ¼ 0.0195). Full details of ANOVA and Dunnett test are providedin Supplementary Table S4. E, Western blot analysis of cleaved PARP and caspase-3 normalized to GAPDH in LNCaP and 22Rv1 cells treated withvehicle control, 10, 20, and 30 mmol/L of SMAP and harvested at 6, 12, and 24 hours. Asterisks, statistical significance based on Dunnett test betweenthe indicated group and the control, where � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ��, P < 0.0001.






approach allowed indirect profiling of proteins that may not bedetected by mass spectroscopy but that are potentially regulatedby SMAP treatment. We were able to score 101 unique kinaseswith at least one identified substrate from our phosphoproteo-mics experiment (Supplementary Tables S1 and S6). Interestingly,

3 of the 4 significantly downregulated kinases (negative score,P < 0.05, 3þ substrates) are documented regulators of AR (Fig.2C): CDK1 (29), CDK5 (30), and SRC (31). Moreover 75% of allsignificantly scored kinases that are known to act on AR (negativeþ positive scores, P < 0.05) had decreased output (Fig. 2D).

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Phosphoproteomics analysis of LNCaP cells with SMAP. A, Volcano plot of the unique phosphosites identified. Horizontal line, P < 0.05 cutoff (calculatedfrom Welch t test between DMSO and SMAP treatment). Vertical lines, 2-fold change cutoffs. The S308 AR phosphosite (purple dot) was found to besignificantly dephosphorylated upon SMAP treatment. B, The top scoring PPI network result from MCODE. Blue nodes, proteins that are dephosphorylatedupon treatment; red nodes, proteins that are hyperphosphorylated upon treatment. Larger circles, proteins with higher degree (number of interactions);circles with dark borders meet a P < 0.05 from Welch test. C, Bar plot summarizing the Kinase Substrate Enrichment Analysis (KSEA) results. Onlykinases with at least three identified substrates are listed. Negative scores indicate kinases with decreased activity output in the treatment group; positivescores are for those with increased activity outputs. Blue (red) bars indicate kinases that meet a P < 0.05 significance cutoff for being significantlydownregulated (upregulated) as calculated from the weighted z-score method employed by Casado and colleagues (28). D, KSEA-calculated kinases withP < 0.05 scores were analyzed for documented interactions to AR. There were four resulting hits, as shown. The majority (3 of the4 kinases) had significantly downregulated output upon treatment, as measured by KSEA (blue circles).

McClinch et al.





Altogether, these findings are consistent with inhibition of manyupstream AR regulators.

As the bioinformatics results converged onto AR signaling,we subsequently decided to probe into SMAP's effects on theprotein itself. Phosphoproteomics revealed significantly lowlevels of the AR-derived S308 phosphopeptide compared tocontrol, with P ¼ 0.028 and -4.1 log2 fold change (Fig. 2A).This particular residue is a previously documented dephos-phorylation site of the catalytic subunit of PP2A (PP2A-C;ref. 5). The phosphorylation status of this site can affect ARtarget gene transcription and AR-mediated cell growth (32).Importantly, the phosphoproteomic pipeline does not mea-sure changes in total protein levels and therefore does notpermit distinction between a decrease in p-AR due to dephos-phorylation versus total AR protein loss. Given this limitation,and in the context of prior studies that demonstrated thatPP2A can promote degradation of its substrates (e.g., MYC),we next probed the effects of SMAP on AR in cell culture.

SMAP decreases AR protein level and disrupts mRNAexpression of multiple AR targets

LNCaP and 22Rv1 cells were treated with 0, 10, 20, and30 mmol/L SMAP and harvested at 1, 3, 6, 12, and 24 hoursafter drug treatment. SMAP induced a time and dose-dependent decrease in AR protein expression in both LNCaP(Fig. 3A and B) and 22Rv1 cells (Fig. 3C) as well as a dose andtime-dependent decrease in PSA protein expression in LNCaPcells (Fig. 3A). Antibodies specific to either the N- or the C-terminal domains of the AR were used for Western blots.Intriguingly, SMAP induced degradation of both AR WT andthe splice variant AR (AR-v7) in 22Rv1 cells. The AR-v7 splicevariant lacks the ligand-binding domain and has been shownto confer resistance to enzalutamide in VCaP and 22Rv1 cells(33). Moreover, the presence of AR-v7 in circulating tumorcells from metastatic CRPC patients correlates with clinicalresistance to enzalutamide and abiraterone (34). AR is regu-lated at multiple levels, with numerous posttranslational mod-ifications (PTM) including phosphorylation, acetylation,sumoylation, ubiquitination, and methylation (35, 36) withAR phosphorylation occupying a predominant role (37). PP2Abinds and dephosphorylates AR at five phosphosites all locatedon the N-terminal domain, including Ser81, Ser94, Ser256,Ser308, and Ser424 (5, 22). Among the most well characterizedof these phosphosites is Ser81, which regulates AR stability,transcriptional activity, and cellular localization (22, 30, 38,39). Given Ser81's role in regulating AR stability as well as itbeing the most stoichemetrically favored phosphosite on theAR (32), we studied its relevance in mediating SMAP-inducedAR degradation. Western blot analysis of LNCaP cells treatedwith 30 mmol/L of SMAP and harvested at 1, 3, 6, 12, and 24hours showed time-dependent Ser81 dephosphorylationoccurring at time-points prior to AR degradation (Fig. 3A andB). In LNCaP cells, AR expression for each time-point evaluatedin Fig. 3A was quantified and the ratio of phosphorylated tototal AR levels was calculated (Fig. 3B; Supplementary TableS7). This result suggests that AR degradation may occur as aresult of PP2A-mediated dephosphorylation of Ser81.Although loss of AR and p-AR appears to begin to occur atthe 10–20 mmol/L dose of SMAP at 6 hours, a dose and time-point where we did not observe pronounced evidence ofapoptosis, the loss of AR and p-AR was most notable at higher

doses of SMAP and/or later time-points. Thus, we cannotcompletely exclude that the observed effects of SMAP on ARand p-AR expression were related to apoptosis.

We next evaluated AR mRNA in dose and time-course stud-ies. qRT-PCR showed that the decrease in AR protein expressionwas not accompanied by a simultaneous decrease in AR mRNAin LNCaP or 22Rv1 cells (Fig. 3D), implying that the decrease inAR levels occurs at the protein level rather than as a conse-quence of a decrease in AR mRNA (e.g., as a result of proteindegradation). To define the downstream effects of SMAP treat-ment on the AR pathway, we next evaluated the transcription ofa panel of AR-regulated genes (AR targets were chosen afterClegg and colleagues; ref. 40) upon SMAP treatment. AR targetgene mRNA expression was measured by qRT-PCR and con-firmed altered expression of multiple AR-regulated genes,including PSA and TMPRSS2 in LNCaP and 22Rv1 cells(Fig. 3D; Supplementary Table S8). These effects occurred ina time-dependent manner, following a pattern consistent withloss of AR transcriptional control. In addition, SMAP treatmentinduced changes in AR target gene expression (Fig. 3D; Sup-plementary Table S8).

SMAP treatment reduces AR half-life in a PP2A-dependentmanner

To determine whether the effects of SMAP on AR proteinwere a result of AR degradation, we next evaluated AR half-life.LNCaP and 22Rv1 cells were preincubated for 3 hours withSMAP or vehicle control before treating with cycloheximide(mg/mL) for 1, 3, 6, and 9 hours to inhibit protein translation.A consistent decrease in AR half-life in LNCaP and 22Rv1 cells(Fig. 4A) was observed in the SMAP-treated samples. To deter-mine whether AR degradation induced by SMAP was protea-some-mediated, LNCaP and 22Rv1 cells were treated for 6hours with vehicle control, 30 mmol/L SMAP, or 30 mmol/LSMAP þ proteasome inhibitor, bortezomib (100 nmol/L forLNCaP and 1 mmol/L for 22Rv1). Treatment with bortezomibimpaired SMAP-induced AR degradation (Fig. 4B; Supplemen-tary Table S9), suggesting that the proteasome pathway wasmediating, in part, SMAP-induced AR degradation. To furtherdefine the role of PP2A in AR degradation, coimmunopreci-pitation of AR protein complexes was performed in LNCaPcells treated with DMSO or 30 mmol/L of SMAP. Significantincreased PP2A binding to the AR after 3 and 6 hours of SMAPtreatment in LNCaP cells was observed by Western blot anal-ysis (Fig. 4C; Supplementary Fig. S4); an IgG-negative controldemonstrated a lack of nonspecific binding of AR or PP2A-C.

To ascertain whether PP2A was mediating SMAP-induced ARdegradation, LNCaP cells were stably transduced with the Simianvirus 40 (SV40) small T antigen (ST), a potent oncoprotein andspecific inhibitor of PP2A (Fig. 5A). The ST alters PP2A's activity byinteracting with the PP2A Aa subunit and displacing regulatory Bsubunits from the dimer. This displacement perturbs the functionof PP2A and its activity toward multiple substrates (41, 42).Overexpression of ST in LNCaP cells resulted in an attenuationof thebiological activity and target engagement of SMAPas shownby Western blot analysis of cleaved PARP, p-AR (Ser81), and AR(Fig. 5A; Supplementary Table S10), providing further evidencethat PP2A is mediating the effects of SMAP on AR degradationanddownstream signaling. As ARposttranslationalmodificationsmay be influenced by ligand binding, and treatment of CRPCoccurs in patients in an androgen-depleted state, we next studied






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SMAP treatment leads to AR protein degradation and changes in AR target genes' mRNA expression in LNCaP and 22Rv1 cells. A, Western blot analysis ofp-AR (ser81), AR (N and C terminus), and PSA normalized to GAPDH in LNCaP cells treated with vehicle control, 10, 20, and 30 mmol/L of SMAPand harvested at 1, 3, 6, 12, and 24 hours. Heatmap of AR protein expression as determined by densitometry of the average fold-change of threeexperimental triplicates. B, Quantified ratio of phosphorylated AR to total AR (average of N- and C-terminus) at 1, 3, 6, 12, and 24 hours after vehiclecontrol, 10, 20, and 20 mmol/L treatment with SMAP. Bars in graph represent fold change of SMAP-treated to vehicle-treated expression. Data aremeans � SD and P values are represented in the graph as � , P < 0.05; �� , P < 0.01. ANOVA, P < 0.0001. Full details of ANOVA and Tukey test are provided inSupplementary Table S7. C, Western blot analysis of AR (N or C terminus) normalized to GAPDH in 22Rv1 cells treated with vehicle control or 10, 20, and30 mmol/L of SMAP and harvested at 1, 3, 6, 12, and 24 hours. Heatmaps of AR protein expression levels as determined by densitometry of theaverage fold-change of three experimental triplicates. D, Heatmaps of mRNA expression levels of AR target genes of LNCaP and 22Rv1 SMAP-treatedcells. Cells were treated with 30 mmol/L SMAP for 6, 12, and 24 hours and mRNA expression was determined by qRT-PCR. AR target genes areseparated into two groups: upregulated AR target genes and downregulated AR target genes. ANOVA P value is provided for each target inSupplementary Table S7. Experiments were performed in triplicate.

McClinch et al.





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SMAP decreases AR half-life in LNCaP and 22Rv1 cells. A, Western blot analysis of AR in LNCaP and AR-WT and AR-V7 in 22Rv1 cells treated with vehiclecontrol and cycloheximide or SMAP (3-hour preincubation) and cycloheximide for 1, 3, 6, and 9 hours. Doses of 30 mmol/L SMAP and 100 mg/mLcycloheximide were used. Plots of AR half- life over a period of 9 hours in LNCaP cells and AR-WT and AR-V7 in 22Rv1 cells in the presence of vehiclecontrol or SMAP. B, Western blot analysis of AR normalized to GAPDH in LNCaP and 22Rv1 cells treated for 6 hours with vehicle control, 30 mmol/L SMAP,bortezomib (100 nmol/L for LNCaP and 1 mmol/L for 22Rv1), or 30 mmol/L SMAP and bortezomib. Western blot densitometry plot of AR relative toGAPDH. PP2A binds to AR after SMAP treatment. The densitometry results depict the averages of three independent experiments � SD. ANOVA wasperformed for LNCaP treated with SMAP (P ¼ 0.0315), 22Rv1 AR WT (P ¼ 0.09), and 22Rv1 ARV7 (P ¼ 0.5349). Full details of ANOVA and Dunnett testare provided in Supplementary Table S9. C, Coimmunoprecipitation of AR protein complexes in LNCaP cells treated with vehicle control or 30 mmol/L ofSMAP for 1 and 3 hours. Western blot analysis showing the coimmunoprecipitation of AR and PP2A-C alone and in the same complex. Western blotdensitometry plot showing the binding fold change of AR with PP2A-C with DMSO and SMAP. The densitometry results depict the averages of threeindependent experiments � SD. � , P < 0.05; �� , P < 0.001.






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PP2A mediates SMAP-induced AR degradation. A,Western blot analysis of phosphorylated AR at Serine 81, AR, cleaved PARP, and small T antigen normalizedto GAPDH in LNCaP cells stably transduced with a retrovirus expressing the small T antigen (LNCaP-ST). LNCaP and LNCaP-ST cells were treated withvehicle control or 30 mmol/L of SMAP for 3 and 6 hours. AR degradation by SMAP is increased in CSS media in LNCaP cells in presence and absenceof R1881. The densitometry results depict the averages of three independent experiments � SD. � , P < 0.05 (ANOVA, P ¼ 0.0031). Full details of ANOVAand Tukey test are provided in Supplementary Table S10. B, Western blot analysis of phosphorylated AR at Serine81 and AR normalized to GAPDH inLNCaP cells treated with vehicle control or 30 mmol/L of SMAP for 3 and 6 hours in FBS or CSS media. Cells that were treated in CSS media were alsotreated with and without 1 nmol/L of R1881 for 3 and 6 hours together with DMSO or SMAP. Western blot densitometry plot of AR relative toGAPDH. The densitometry results depict the averages of three independent experiments using � SD. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.ANOVA was performed for LNCaP at 3 hours (P < 0.0001) and 6 hours (P < 0.0001). Full details of ANOVA and Tukey test are provided in SupplementaryTable S11. C, Heatmaps of AR target gene mRNA expression levels in LNCaP cells treated with vehicle control or 30 mmol/L of SMAP for 3 and 6 hoursin FBS or CSS media. AR target genes are separated into two groups: upregulated AR target genes and downregulated AR target genes. Cells thatwere treated in CSS media were also treated with and without 1 nmol/L of R1881 for 3 and 6 hours together with DMSO or SMAP. P values werecalculated for each individual gene using ANOVA (Supplementary Table S12).

McClinch et al.





SMAP's ability to degrade AR protein (Fig. 5B; SupplementaryTable S11) and mRNA (Fig. 5C; Supplementary Table S12) inLNCaP cells grown in charcoal-stripped FBS (CSS) media and inthe presence of an AR agonist (R1881). AR transcriptional andtranslational degradation was enhanced in LNCaP cells grown inCSSwith 30mmol/L of SMAPwhen comparedwith LNCaP cells inFBS media with 30 mmol/L of SMAP, for 3 and 6 hours at themRNAandprotein level (Fig. 5B andC). This enhancedARmRNAand AR protein degradation in the CSS media was not overcomeby the addition of 1 nmol/L of R1881 to SMAP-treated cells (Fig.5B and C).

SMAPs inhibit tumor growth in a LNCaP/AR xenograftmodel

To evaluate the antitumor activity of SMAP as a single agentin vivo, we used the LNCaP-AR xenograft (parental LNCaP cellsoverexpressing WT-AR to model the clinical scenario; ref. 43)model in SCID mice. MDV3100 (enzalutamide) was utilized asa comparator in these studies to assess SMAP activity relativeto a current standard of care in the disease. Mice were dosed for38 days in the following treatment groups: vehicle control (n ¼6), 100 mg/kg SMAP twice daily (n ¼ 6), 400 mg/kg SMAPtwice daily (n ¼ 6), and 100 mg/kg MDV3100 once daily (n ¼5). Treatment in all groups was orally administered. Two dosesof SMAP were used to assess whether there was a relationshipbetween dose and antitumor activity. SMAP delivered at 400mg/kg twice daily resulted in durable stasis (Fig. 6A; Supple-mentary Table S13) and mice were assessed for signs of toxicityevery other day for the duration of the study. No significantweight loss or signs of toxicity (i.e., diarrhea, abdominalstiffness, lethargy) were noted (Fig. 6B). The antitumor activityof the 400 mg/kg SMAP treatment group was comparable withenzalutamide (Fig. 6A–C; Supplementary Table S13). In par-allel drug development efforts, a second-generation compoundwas prepared that exhibited greater potency than SMAP. Thesecompounds, exemplified by SMAP-2, contain a cyclic linkervariation (Supplementary Fig. S2; ref. 19). It should be notedthat restricting rotatable bonds can increase bioavailability andon-target potency while decreasing off-target effects. Exhibitingsimilar bioavailability to SMAP, SMAP-2 demonstratedincreased potency in several cell lines (Supplementary Fig.S3) prompting its evaluation in mouse models of CRPC.SMAP-2 was tested in two in vivo efficacy studies in bothcastrated and noncastrated models of prostate cancer in SCIDmice harboring LNCaP/AR xenograft tumors. In the nonca-strated efficacy study, mice were randomized to two treatmentgroups based on initial tumor volumes: Control (n ¼ 11) orSMAP-2 100 mg/kg twice daily (n ¼ 8) and treated for 28 days.Tumor volumes and body weights were measured every otherday. SMAP-2 demonstrated significant activity as defined byfold change in tumor volume (final tumor volume/initialtumor volume) between the control and SMAP-2 (7.4 vs. 3.6;�, P< 0.01) treated groups without significant weight loss in thedrug-treated mice compared with vehicle control–treated miceafter one month of dosing (Fig. 6D–F). In the castration study,which was utilized to mimic the clinical scenario in whichCRPC patients receive treatment in an androgen-depleted state,male SCID mice were castrated 2 weeks before beginningtreatment on either vehicle control (n ¼ 11) or 100 mg/kgSMAP-2 twice daily (n ¼ 9). The SMAP-2 group demonstratedsignificant activity as defined by fold change in tumor volume

as compared with control-treated group (1.4 vs. 6.7, respec-tively; ��, P < 0.0001; Fig. 6G–I). These studies further estab-lished the safety and efficacy of SMAP-2 in prostate cancerin vivo models; no significant weight loss (Fig. 6H), clinicalchemistry abnormalities, or behavioral signs were observed inthe drug-treated mice after one month of dosing (Fig. 6B).

Upon establishing the safety and efficacy of SMAP-2 in theLNCaP/AR model in vivo, we subsequently explored the phar-macodynamic effects of treatment. Castrated LNCaP/AR tumor-bearing mice were treated for 6 days with SMAP-2, at 30 mg/kg(n ¼ 7) or 100 mg/kg twice daily (n ¼ 8), and vehicle control(n ¼ 12). Remarkably, when the drug formulation wasswitched from suspension to solution SMAP-2 demonstratedsignificant activity (Supplementary Fig. S5A). Tumor regres-sions and no significant weight loss was observed in both the30 mg/kg and 100 mg/kg SMAP-2 treatment groups afteronly 6 days of treatment (Fig. 7A–C; Supplementary TableS14), and greater antitumor activity correlated with betterexposure in serum analyzed from drug-treated mice (Supple-mentary Fig. S5B and S5C). Posttreatment serum chemistrypanels displayed no adverse effects on liver, kidney, or pan-creatic function in SMAP-2–treated mice. Moreover, SMAP-2treatment decreased proliferation and induced apoptosis asdetermined by PCNA and TUNEL staining of tumor samplesfrom drug-treated animals, respectively (Fig. 7D–F; Supple-mentary Tables S15 and S16). Effects on tumor volume corre-lated strongly with downstream markers of target engagementin vivo as measured by Western blot analysis of AR and PSAprotein expression in representative tumor samples (Fig. 7 G–I;Supplementary Tables S17 and S18).

DiscussionDespite an expanded armamentarium over the past decade

for the treatment of CRPC driven by a better understanding ofthe underlying pathogenesis of the disease, resistance devel-ops to all available therapies contributing to over 27,000deaths each year in the United States. The AR, including splicevariant forms lacking the ligand-binding domain, remains acentral therapeutic target in treatment-resistant CRPC thoughseveral additional targets have been identified. Novelapproaches capable of eliminating AR signaling, while simul-taneously inhibiting complimentary oncogenic signaling net-works, are critically needed to further improve the outcomesof men with CRPC.

Protein phosphatase 2A is a bona fide tumor suppressor and isimplicated specifically in the pathogenesis of CRPC (14). Indeed,dephosphorylation of key oncogenic proteins, such as MYC,AKT1, BCL2, and AR likely explains the critical role of PP2A inprostate cancer. Unlike the majority of tumor suppressors, whichare inactivated in cancer due to loss-of-function genetic muta-tions, mutations of components of the PP2A heterotrimer arerelatively rare in human malignancy, with the exception of endo-metrial cancer. Rather, PP2A function is most commonlydecreased as a consequence of decreased subunit expressionand/or overexpression of endogenous inhibitors such as CIP2Aand SET raising the possibility of therapeutic activation as a noveltreatment strategy. Consistent with this hypothesis, knockdownof SET has recently demonstrated anticancer effects in enzaluta-mide-resistant prostate cancer cell lines and mouse models (44).Protein phosphatases themselves, however, have been largely






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SMAP and SMAP-2 inhibit tumor growth in a LNCaP/AR xenograft model in both castrated and noncastrated male mice. A, Fold change in tumor volume wasassessed every 2–3 days in noncastrated male mice, bearing on average 200 mm3 LNCaP/AR tumors, treated with vehicle control (n ¼ 6), 100 mg/kgSMAP twice daily (n ¼ 6), 400 mg/kg SMAP twice daily (n ¼ 6), or 100 mg/kg MDV3100 (n ¼ 5) once daily for 38 days. Data shown represents themean tumor volume � SD. ANOVA, P ¼ 0.0004. B, Fold change in body weight of mice over the course of the SMAP treatment study. C, Waterfall plot ofpercent change in individual tumor volume from day 0 to 38 for each mouse in SMAP treatment study. ANOVA, P ¼ 0.0246. Full details of ANOVA andDunnett tests are provided in Supplementary Table S13. D, Fold change in tumor volume was assessed every 2–3 days in noncastrated male micebearing LNCaP/AR tumors, on average 100 mm3, treated with vehicle control (n ¼ 11) or 100 mg/kg SMAP-2 (n ¼ 8) orally twice daily for 28 days. E, Foldchange in body weight of mice over the course of the SMAP-2 treatment study. F, Waterfall plot of percent change in individual tumor volumes fromday 0–28 for each mouse in SMAP-2 treatment study. G, Fold change in tumor volume assessed every 2–3 days in castrated male mice bearing LNCaP/ARtumors, on average 200–250 mm3, treated with control (n ¼ 11) or 100 mg/kg SMAP-2 (n ¼ 9) orally twice daily for 31 days. H, Fold change in bodyweight of castrated mice over the course of the SMAP-2 treatment study. I, Waterfall plot of percent change in individual tumor volume from day 0to 31 for each mouse in SMAP-2 treatment study. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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SMAP-2 inhibits tumor growth and reduces AR and PSA expression in vivo in a pharmacodynamic study in a castrated male LNCaP/AR xenograft mousemodel. A total of 5 � 106 LNCaP/AR cells was subcutaneously injected into the right flank of castrated SCID mice and allowed to grow to an averageof 200 mm3. Mice were treated twice daily with vehicle control, 30 mg/kg SMAP-2, and 100 mg/kg SMAP-2 for 6 days. SMAP-2 was administered orally in ahomogenous solution comprised of N,N-Dimethylacetamide, Solutol, and water. A, Fold change of tumor volume assessed every 2 days for the durationof the study. B, Fold change in body weight of mice over the course of the pharmacodynamic study. C, Waterfall plot of percent change in individualtumor volumes from day 0–6, ANOVA, P ¼ 0.0029. Full details of ANOVA and Dunnett test are provided in Supplementary Table S14. D, Representativemicroscopy images of treated and control xenograft tumor sections resected 2 hours after final dose and stained for TUNEL and PCNA. Quantificationof the percentage of TUNEL-positive cells (green; ANOVA, P ¼ 0.0289) and PCNA-positive cells (brown; ANOVA, P < 0.0001) in treated and control xenografttumors is shown in E and F, respectively. Full details of ANOVA and Dunnett are provided in Supplementary Tables S15 and S16. G, Representativeimmunoblots of AR and PSA protein expression in control and treated xenograft tumors resected 2 hours after final dose as performed by immunoblottingand densitometry. Quantification of AR (H) and PSA (I) protein expression in control and treated xenograft tumors resected 2 hours after final dose asperformed by immunoblotting and densitometry. ANOVA was performed for AR (P ¼ 0.0199) and PSA (P ¼ 0.0075). Full details of ANOVA and Dunnett testsare provided in Supplementary Tables S17 and S18. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






ignored for drugdevelopment because of their perceived "undrug-gable" nature. We have developed a novel class of molecules,which directly bind PP2A, promote conformational changes, andthereby lead to phosphatase activation (19).

Here, we show that our SMAPs induce anticancer effects inmodel systems of prostate cancer while modulating known PP2Asubstrates. In cell culture and in vivo models of prostate cancer,SMAP treatment led to induction of apoptosis. Given the largenumber of known PP2A substrates important in oncogenesis, weemployed a global phosphoproteomic approach to begin toprobe the mechanistic basis for the observed anticancer effects.

These studies revealed that the AR was among the most signif-icantly dephosphorylated proteins by SMAP treatment and thatmembers of its signaling axis were downregulated. We focusedsubsequent experiments on AR, given its central role in CRPC,demonstrating that SMAP induces dephosphorylation, and pro-teasome-mediated degradation, of the AR (including the splicevariant AR-v7) in cell culture. The effects of our SMAP on the ARwere confirmed to be PP2A dependent, as expression of the STabrogated these effects. Finally, a decrease in AR protein expres-sion correlated with the growth-inhibitory effects of SMAPs inprostate cancer xenograftmodels. Thedevelopment of therapeuticapproaches capable of degrading full-length and splice variant ARcould be potentially transformative in the treatment of CRPC.

The large number of possible posttranslationalmodifications ofthe AR, including phosphorylation/dephosphorylation of severaldistinct phosphosites, has complicated an understanding of theglobal role of these events and, to date, precluded their therapeuticexploitation. Here we show that SMAP treatment results in deg-radation. Indeed, phosphorylation of this site has previously beenshown to regulate AR stability, transcriptional activity, and cellularlocalization (22, 30, 38, 39). Importantly, all of the known PP2A-ARphosphosites are localized to theN-terminal domain of theAR,potentially explaining the degradation of both full-length andsplice variant AR observed in cell culture with SMAP treatment(36). Although the role of SMAP-mediated AR dephosphorylationin inducing AR degradation is supported by our time-coursestudies and prior studies exploring the functional consequencesof AR phosphorylation, the mechanism by which these eventstarget the AR for proteasome-mediated degradation, and thespecific phosphosites involved, require further investigation.

In addition, despite the critical role of the AR in driving prostatecancer growth, further studies are required to establish the directlinkbetweenARdegradation and the anticancer effects inducedbySMAPs. Studies employing AR phosphosite mutants are currentlyplanned to shed additional light on these issues. Indeed, SMAPsmay also serve as important tools to advance our general under-standing of the functional consequences of posttranslationalmodifications of the AR.

There are potential limitations to our study. Our phosphopro-teomic studies involved a single cell line and a single time-point.The optimal time-point for phosphoproteomic analysis wasdetermined from time course studies performed in cell cultureto identify a time at which reproducible changes were detected inphosphorylation yet prior to induction of apoptosis. Thisapproach facilitated the determination of direct drug-mediatedphosphorylation changes while limiting the potential secondarychanges in the phosphoproteome resulting from the induction ofcell death. In spite of this, it is possible that the PP2A substratesidentified by such analyses are dependent on the particular PP2Aregulatory (B) subunits expressed by a cell line and the timing of

the binding and dephosphorylation events. Furthermore, thephosphoproteomic pipeline does not permit distinction betweenloss of p-AR due to dephosphorylation versus decrease in total ARexpression. Still, these studies did identify AR as among the mostsignificantly affected proteins. Decreased p-AR and AR proteinexpression was observed with SMAP treatment in a dose- andtime-dependent manner in LNCaP and 22Rv1 cells; given theinduction of apoptosis observedwith SMAP treatment, we cannotexclude that the loss of p-AR andARwas related to cell death ratherthan dephosphorylation and degradation though additionalstudies with cycloheximide or bortezomib were supportive ofthe latter. Demonstration of target engagement in our in vivostudies focused on events downstream of PP2A, specifically ARand PSA. While one might consider functional assays of PP2A intumor or tissue extracts as a means to demonstrate target engage-ment, such an approach has multiple limitations that precludeaccurate and reproducible results including (i) the need to sep-arate and recover PP2A from other phosphatases given the lack ofspecificity of phosphatase assays and the (ii) high probably ofdissociation of SMAPs from PP2A during preparation of extractsgiven noncovalent bonding.

However, our prior data demonstrating that our SMAPsdirectly bind and activate PP2A (19), coupled with our currentstudy demonstrating that PP2A inhibition (with small T anti-gen) abrogated the AR-degrading effects of these molecules incell culture, does provide support that the pharmacodynamiceffects observed in vivo were indeed a result of PP2A activation.The activity with SMAPs in cell culture experiments occurs in themicromolar range as compared with kinase inhibitors, whichtend to exert activity in the nanomolar range. There are severalreasons for these differences in relative potency. PP2A is a veryabundant cellular enzyme with an intracellular concentrationof approximately 250 nmol/L and the cell-free–binding affinityof these SMAPs at PP2A A-alpha is 235nmol/L and the stoichi-ometry of binding is 1:1 (19). Given that protein binding forthese compounds is approximately 96%, the free drug concen-tration (which based upon our studies drives the drug activity) isapproximately 1–2 mmol/L for a 10-mmol/L drug dose in 10%serum containing medium. Thus, given the mechanism of action(allosteric agonism), the abundance of the cellular target PP2A,and the protein binding for this drug series, cellular potencies ofless than 1 mmol/L are most likely not achievable.

Importantly, PP2A substrates other than the AR may be con-tributing to the anticancer effects observed with SMAP treatment,including MYC. MYC is one of the best characterized PP2Asubstrates and PP2A has previously been shown to prime MYCfor proteasome-mediated degradation through dephosphoryla-tion of serine 62 (45). In breast cancer models, depletion orinhibition of the endogenous PP2A inhibitors, CIP2A or SET,reduced MYC expression and activity and decreased tumorigenicpotential (46). Chromosome 8q, including the MYC gene, isamplified in approximately 30% of human prostate cancers andMYC overexpression recapitulates human prostate carcinogenesisin genetically engineered mouse models (47, 48). Prior reportshave shown that MYC is further upregulated in CRPC and con-tributes to independent prostate cancer cell growth (22–25).Furthermore, MYC has been shown to be a key androgenligand-independent AR target gene and MYC depletion was dem-onstrated to reduce prostate cancer cell survival in androgenligand-depleted conditions (49). Together, these findings raisethe hypothesis that simultaneously cotargeting AR and MYC for

McClinch et al.





degradation is responsible for the anticancer effects observedwithSMAPs in prostate cancer model systems. Additional studies areplanned to define the effect onMYC induced by SMAP treatment.Similarly, the role of other known PP2A substrates previouslyimplicated in CRPC pathogenesis, including AKT1, BCL2, andothers warrant further investigation.

Disclosure of Potential Conflicts of InterestD.B. Kastrinsky has ownership interest (including patents) in Dual Thera-

peutics. D.L. Brautigan reports receiving a commercial research grant fromBristol–Myers Squibb and is a consultant/advisory board member for DualTherapeutics and Bristol-Myers Squibb. M. Ohlmeyer is a consultant atDual Therapeutics LLC, reports receiving a commercial research grant fromDual Therapeutics LLC, has ownership interest (including patents) in DualTherapeutics, and is a consultant/advisory boardmember forDual TherapeuticsLLC. M.D. Galsky has ownership interest (including patents) in Dual Thera-peutics. The Icahn School ofMedicine atMount Sinai, on behalf of G. Narla andM. Ohlmeyer, has filed patents covering composition of matter on the smallmolecules disclosed herein for the treatment of human cancer and otherdiseases (International Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2). RAPPTA Therapeutics LLC haslicensed this intellectual property for the clinical and commercial developmentof this series of small-molecule PP2A activators. G. Narla, M. Ohlmeyer, andM.D. Galsky have an ownership interest in RAPPTA Therapeutics LLC. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K. McClinch, R.A. Avelar, D. Callejas, M. Cooper,D. McQuaid, A.C. Levine, M. Ohlmeyer, G. Narla, M.D. GalskyDevelopment of methodology: K. McClinch, R.A. Avelar, D. Callejas,S. Izadmehr, J. Sangodkar, D. Schlatzer, M. Cooper, R.C. Sears, M. Ohlmeyer,G. Narla, M.D. GalskyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. McClinch, R.A. Avelar, S. Izadmehr, A. Perl,

J. Sangodkar, D. Schlatzer, M. Cooper, J. Kiselar, A. Stachnik, S. Yao, D. Hoon,S.R. Plymate, A.C. Levine, A. DiFeo, G. Narla, M.D. GalskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. McClinch, R.A. Avelar, D. Callejas, S. Izadmehr,D. Wiredja, A. Perl, J. Sangodkar, D. Schlatzer, M. Cooper, A. Stachnik,S.R. Plymate, A.C. Levine, A. Kirschenbaum, J.P. Sfakianos, G.Narla,M.D. GalskyWriting, review, and/or revision of the manuscript: K. McClinch, R.A. Avelar,S. Izadmehr, D. Wiredja, A. Perl, J. Sangodkar, D.B. Kastrinsky, M. Cooper,A. Stachnik, D. McQuaid, D.L. Brautigan, S.R. Plymate, C.C.T. Sprenger,W.K. Oh, A.C. Levine, J.P. Sfakianos, A. DiFeo, G. Narla, M.D. GalskyAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): K. McClinch, S. Izadmehr, J. Sangodkar,D.B. Kastrinsky, S. Yao, Y. Gong, G. Narla, M.D. GalskyStudy supervision: R.A. Avelar, Y.A. Ioannou, G. Narla, M.D. GalskyOther [involved in chemical synthesis to generate small-molecule activa-tors of protein phosphatase-2A (SMAP)]: N. ZawareOther (design and synthesis of small-molecule PP2A activators that are thesubject of paper): M. Ohlmeyer

AcknowledgmentsWe gratefully acknowledge the New York City Investment Fund (Bio-

accelerate Prize) and Dual Therapeutics/BioMotiv for continued support.G. Narla received a Howard Hughes Medical Institute (HHMI) Physician-Scientist Early Career Award and a Case Comprehensive Cancer CenterPilot Award. S. Izadmehr is a LRP awardee (NCATS/NIH). This workwas partially supported by an NCI/NIH R01 grant 1R01CA181654-01A1(G. Narla, M.D. Galsky, M. Ohlmeyer, and A.C. Levine), DoD grantW81XWH-15-1-0596 (M.D. Galsky), and Prostate Cancer FoundationYoung Investigator Award (M.D. Galsky).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 13, 2017; revisedMay 13, 2017; accepted January 17, 2018;published online January 22, 2018.

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2018;78:2065-2080. Published OnlineFirst January 22, 2018.Cancer Res Kimberly McClinch, Rita A. Avelar, David Callejas, et al. Treatment of Castration-Resistant Prostate CancerSmall-Molecule Activators of Protein Phosphatase 2A for the

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