Anagrelide for Gastrointestinal Stromal Tumor · Translational Cancer Mechanisms and Therapy...

Translational Cancer Mechanisms and Therapy

Anagrelide for Gastrointestinal Stromal TumorOlli-Pekka Pulkka1, Yemarshet K. Gebreyohannes2, Agnieszka Wozniak2,John-Patrick Mpindi3, Olli Tynninen4, Katherine Icay5, Alejandra Cervera5,Salla Keskitalo6, Astrid Murum€agi3, Evgeny Kulesskiy3, Maria Laaksonen7,Krister Wennerberg3, Markku Varjosalo6, Pirjo Laakkonen8, Rainer Lehtonen5,Sampsa Hautaniemi5, Olli Kallioniemi3, Patrick Sch€offski2, Harri Sihto1, andHeikki Joensuu1,9

Abstract

Purpose: Gastrointestinal stromal tumor (GIST) is a com-mon type of soft-tissue sarcoma. Imatinib, an inhibitor of KIT,platelet-derived growth factor receptor alpha (PDGFRA), and afew other tyrosine kinases, is highly effective for GIST, butadvanced GISTs frequently progress on imatinib and otherapproved tyrosine kinase inhibitors. We investigated phos-phodiesterase 3 (PDE3) as a potential therapeutic target inGIST cell lines and xenograft models.

ExperimentalDesign: TheGIST gene expression profile wasinterrogated in the MediSapiens IST Online transcriptomedatabase comprising human tissue and cancer samples, andPDE3A andPDE3B expressionwas studied using IHCon tissuemicroarrays (TMA) consisting of 630 formalin-fixed humantissue samples. GIST cell lines were screened for sensitivity to217 anticancer compounds, and the efficacy of PDE inhibitors

on GIST was further studied in GIST cell lines and patient-derived mouse xenograft models.

Results: GISTs expressed PDE3A and PDE3B frequentlycompared with other human normal or cancerous tissuesboth in the in silico database and the TMAs. Anagrelidewas identified as the most potent of the PDE3 modulatorsevaluated. It reduced cell viability, promoted cell death,and influenced cell signaling in GIST cell lines. Anagrelideinhibited tumor growth in GIST xenograft mouse models.Anagrelide was also effective in aGIST xenograftmousemodelwith KIT exon 9 mutation that may pose a therapeutic chal-lenge, as these GISTs require a high daily dose of imatinib.

Conclusions: PDE3A and PDE3B are frequently expressedin GIST. Anagrelide had anticancer efficacy in GIST xenograftmodels and warrants further testing in clinical trials.

IntroductionGastrointestinal stromal tumor (GIST) is the most common

type of sarcoma that arises from the gastrointestinal tract. GISTsfrequently harbor aberrant KIT or platelet-derived growth factorreceptor alpha (PDGFRA) that encode constitutively activatedreceptor tyrosine kinases (1, 2), but a minority of GISTs lack a

mutation in these genes (3). Most GIST patients with a KITmutation or a PDGFRA mutation can be effectively treated withtyrosine kinase inhibitors such as imatinib or other agents (4), butsecond mutations conferring drug resistance emerge frequentlyleading to GIST progression (5). Sunitinib and regorafenib, theapproved second-line and third-line agents for advancedGIST, areeffective, but the median time to disease progression was 6months or less with these agents in the pivotal randomized trials(6, 7). Therefore, there is a need for novel effective agents for thetreatment of patients with advanced GIST.

Phosphodiesterase (PDE) 3A and 3B are cyclic nucleotidephosphodiesterases that regulate the intracellular concentration,localization, and signaling of cyclic AMP (cAMP), and/or cyclicGMP (cGMP) by controlling their degradation (8). cAMP andcGMP signalings regulate various physiologic processes, includ-ing cell proliferation and differentiation, inflammation, geneexpression, apoptosis, and metabolic pathways (9–11). PDE3Ais expressed in the smooth muscle, platelets, and cardiac tissues,and it is involved in platelet aggregation and in the regulation ofthe blood pressure, cardiac function, and oocyte meiosis (8, 12).PDE3B is frequently expressed in cells that are important in energyhomeostasis, such as adipocytes and hepatocytes (8). PDE3B isimportant especially in insulin signaling (13). Some types ofcancer, such as chronic lymphocytic leukemia (CLL) and colo-rectal cancer, express low levels of cAMP as a consequence ofoverexpression of PDEs (14, 15), and inhibition of various PDEshas antitumor effects in several cancer cell lines (16).High PDE3Aexpression is associated with Sclaffen12 (SLFN12) expression inGIST (17, 18). In one study, the PDE3 inhibitor cilostazol reduced

1Laboratory of Molecular Oncology, Research Programs Unit, TranslationalCancer Biology, Department of Oncology, University of Helsinki, Helsinki,Finland. 2Laboratory of Experimental Oncology, Department of Oncology, KULeuven and Department of General Medical Oncology, University HospitalsLeuven, Leuven, Belgium. 3Institute for Molecular Medicine Finland (FIMM),University of Helsinki, Helsinki, Finland. 4Department of Pathology, HaartmanInstitute, University of Helsinki and HUSLAB, Helsinki, Finland. 5ResearchPrograms Unit, Genome-Scale Biology, Medicum and Department of Biochem-istry and Developmental Biology, Faculty of Medicine, University of Helsinki,Helsinki, Finland. 6Institute of Biotechnology, University of Helsinki, Helsinki,Finland. 7MediSapiens Ltd., Helsinki, Finland. 8Research Programs Unit, Trans-lational CancerBiology, University of Helsinki, Helsinki, Finland. 9ComprehensiveCancer Center, Helsinki University Hospital, Helsinki, Finland.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H. Sihto and H. Joensuu share authorship of this article.

Corresponding Author: Heikki Joensuu, Helsinki University Hospital and Uni-versity of Helsinki, Haartmaninkatu 4, FIN-00029 Helsinki, Finland. Phone: 358-9-471-73208; Fax: 358-9-471-74202; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0815

�2018 American Association for Cancer Research.

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theGIST882 cell line viability, and the PDE3A SLFN12 interactionwas suggested to have a role in cell death (17).

Anagrelide hydrochloride targets the PDE3 enzyme family andreduces the peripheral blood platelet numbers by inhibitingmegakaryopoiesis in the bone marrow (19, 20), and is used forthe treatment of essential thrombocythemia (21, 22). Anagrelideinhibits the cAMP phosphodiesterase activity elevating the cAMPlevels in platelets (23, 24).

In the present study, we investigated the role of PDE3 in GIST.We found that both PDE3A and PDE3B enzyme isoforms arefrequently highly expressed inGISTs,modulationofPDE3activitywithout SLFN12 coexpression decreased the GIST cell prolifera-tion rate in cell culture and promoted apoptosis, and a PDE3inhibitor, anagrelide, inhibited GIST growth in patient-derivedmouse xenograft models.

Materials and MethodsTissue samples

A total of 630 formalin-fixed, paraffin-embedded (FFPE) tumorsamples were retrieved from the archives of the Department ofPathology, Helsinki University Hospital, and studied for KIT,ANO1 (anoctamin-1, DOG-1), PDE3A, and PDE3B expressionusing IHC. The series consists of 36 different histologic types ofhuman cancer and included 55 GISTs (Table 1). The GISTs hadbeen diagnosed by a pathologist from a tissue sample, each hadmorphology compatible with GIST, and each expressed KIT and/or ANO1 at IHC. We selected 7 GISTs and 30 leiomyosarcomaswith sufficient tumor tissue available out of these 630 samples forqPCR analyses of PDE3A and PDE3B mRNA. The InstitutionalReview Board of theHelsinki University Hospital and the Nation-al Supervisory Authority for Welfare and Health, Finland,approved the study (permissions HUS 291/13/03/02/2011 and377/06.01.03.01/2012, respectively).

Gene tissue index analysisThe gene tissue index (GTI) is amodified version of the poverty

index formula used in economics (25). The GTI can be used todetermine the proportion of outlying samples within a disease

group relative to a reference group and for identifying the geneswith outlier expression. A large positiveGTI for a gene indicates anoutlier in the disease group and a large negative GTI an outlier inthe control group. The standard statistical outlier cutoff per gene isdetermined considering all samples.

The GTI analysis was done by comparing RNA expression ofeach gene in 77 GIST samples against all other tissues withexpression data available in theMediSapiens ISTOnline database(http://ist.medisapiens.com/). In these analyses, the number ofreference samples varied from 10,654 to 19,986 and the numberof reference tissue types from 317 to 409. The results were filteredusing identifiedmammalian drug target genes from https://www.drugbank.ca (26). These analyses that provided a list of top 25drug target genes with the largest GTI values had a minimum of16,436 reference samples from 389 tissues.

High-throughput drug screeningThe drug library contained 217 approved or investigational

anticancer compounds. Seventeen drugs were excluded from theanalyses due to technical problems. The high-throughput drugsensitivity and resistance testing was performed as previouslydescribed (27). In brief, the compounds were first dissolved inDMSOand plated at 5 different concentrations covering a 10,000-fold concentration range on 384-well plates. After this, GIST882(1,000 cells/well) and GIST48 cells (2,000 cells/well) were platedon the 384-well plates. Cell viability was measured after 72-hourincubation at þ37�C by using the CellTiter-Glo Cell ViabilityAssay (Promega Inc.) and a PHERAstar FS plate reader (BMGLabtech). The data were normalized to negative (DMSO only)and positive (100 mmol/L benzethonium chloride) controls.The Marquardt–Levenberg algorithm was used to estimate thefour-parameter logistic dose–response curves using the internallydeveloped Breeze analysis platform (28). Drug responses to thetest compounds were measured using the drug sensitivityscore (DSS) that integrates the multiparametric dose–responserelationships between cancer cells and control cells into a singlemetric (28). A high DDS indicates high responsiveness of thecells to a drug.

Cell linesThe GIST cell lines GIST882 and GIST48 were kindly provided

by Dr. Jonathan A. Fletcher (Harvard Medical School, Boston,MA). GIST882 harbored a homozygousmissensemutation inKITexon 13 encoding a p.K642E-mutant oncoprotein, and GIST48 ahomozygous KIT exon 11missensemutation leading to p.V560Dand a heterozygous secondary exon 17 (kinase activation loop)missense mutation leading to p.D820A with an allele frequencyof 25% in next-generation sequencing using an Illumina HiSeq2000 sequencer (Illumina). TheGIST cell lines weremycoplasma-tested. The cells were cultured in a humidified 5% CO2 atmo-sphere at 37�C. The GIST cell lines were cultured in a RPMI 1640medium (GIBCO) supplemented with 20% FBS with 2% peni-cillin/streptomycin (GIBCO).

PDE inhibitors and imatinibCilostazol, milrinone, and amrinone were purchased from

Sigma, and anagrelide from Lancrix. Imatinib used for the cellculture experimentswas purchased fromCaymanChemical Com-pany, and imatinib used for the mouse xenograft experimentsfrom Sequoia Research Products Ltd. Imatinib was reconstitutedin water. All other inhibitors were reconstituted in DMSO. For the

Translational Relevance

Gastrointestinal stromal tumors (GIST) frequently harboreithermutated KIT or platelet-derived growth factor receptor alpha(PDGFRA) that encode a constitutively activated receptortyrosine kinase. Imatinib, an inhibitor of KIT, PDGFRA, anda few other kinases, revolutionized the systemic treatment ofGIST, but second mutations that inhibit imatinib and othertyrosine kinase inhibitors from binding to the activatedkinases often eventually emerge leading to GIST progression.Enzymes of the phosphodiesterase 3 family (PDE3A andPDE3B) are highly expressed in GIST compared with manyother types of human cancer, whichmay open an opportunityfor an efficient targeted therapy. Anagrelide, a PDE3-specificmodulator marketed for the treatment of thrombocythemia,decreases GIST cell proliferation and promotes their apoptosisin vitro. Anagrelide inhibited GIST growth in patient-derivedmouse xenograft models. Anagrelide may have therapeuticvalue in the treatment of GIST.

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xenograft study, anagrelide was administered as a suspension in10% ethanol.

cAMP and cGMP assaysPDE3 activity changes in the GIST882 and GIST48 cell lines

were analyzed indirectly by measuring the amount of cAMP andcGMP. Cells were treated with cilostazol, milrinone, amrinone, oranagrelide hydrochloride for 6 hours and lysed in 0.1 mol/L HCl,and the concentrations of cAMP and cGMP were determinedusing a cAMP Enzyme Immunoassay kit or a cGMP EnzymeImmunoassay kit (SIGMA). cAMP and cGMP changes were mea-sured in two separate experiments (in each, n ¼ 6).

GIST xenograft modelsThe patient-derived GIST models were established from con-

sented patients, and xenografting was approved by the Medical

Ethics Committee, University Hospitals Leuven (Leuven,Belgium). The in vivo experiments were approved by the EthicsCommittee for Animal Research, KU Leuven (Leuven, Belgium),and were conducted according to their guidelines and the Belgianregulations.

Four GIST xenograft mouse models were generated. Threemodels were patient-derived, and one was based on the GIST882cell line. Two models were considered imatinib-sensitive, theGIST882model (with KIT exon 13mutation leading to p.K642E)and the UZLX-GIST3 model (with KIT exon 11 mutation leadingto p.W557_V559delinsF). The UZLX-GIST2B model was consid-ered dose dependently imatinib-sensitive (with KIT exon 9muta-tion leading to p.A502_Y503dup), and the UZLX-GIST9 modelimatinib-resistant (with KIT exon 11 mutations leading top.P577del and W557LfsX5, and a secondary KIT exon 17 muta-tion leading to p.D820G).

Adult female athymic Naval Medical Research Institute mice(NMRI nu/nu, Janvier Laboratories) were transplanted bilaterallywith the GIST cells as described previously (29). Eachmouse was10 to 12 weeks old at the time of the transplantation. The micewere randomly assigned to the treatment groups. The investiga-tors were not blinded during experiments and the outcomeassessment. Two control mice cohorts were used, one treatedwith 10% ethanol (the vehicle for anagrelide, 5 mg/kg/bid) andone with imatinib (100 mg/kg/qd). The daily imatinib dose of100 mg/kg is close to the human equivalent of 400 mg per day.The test mice were treated with anagrelide (5 mg/kg/bid) or withthe combination of anagrelide and imatinib (given at the samedose and schedule as the single agents). The effective anagrelidedosage range was searched in a dose-finding study using theGIST882 xenograft mouse model, where the anagrelide dose wasincreased stepwise until an effective dose level was reached.Treatment efficacy in the anagrelide group was compared withthat of the vehicle group and the imatinib group. Each groupconsisted of 4 to 6 mice. The tumor volumes (measured 3 timesper week) and the mice body weight (measured daily) wereassessed for 10 to 29 days until the mice were sacrificed (or whenthe tumor diameter exceeded2 cm, the loss of thebodyweightwas>20%, or for another ethical reason).

Both FFPE tissue samples and frozen tissue samples werecollected from the sacrificed mice for molecular analyses. TheFFPE tumor specimens were cut into 4-mm sections for stainingwith hematoxylin and eosin (H&E) and for IHC. The proportionsof tumor necrosis, myxoid degeneration, and/or fibrosis wereestimated on the H&E-stained slides, and graded either as grade 1(0% to 10%), grade 2 (>10% and �50%), grade 3 (>50% and�90%), or grade 4 (>90%; ref. 30).

mRNA and miRNA sequencingGIST882 and GIST48 cells were plated at a density of

900,000 cells on 6-well plates. After 24 hours, GIST cells weretreated with 10 mmol/L anagrelide hydrochloride. For mRNAand miRNA sequencing, the cells were scraped into a lysisbuffer at time points 0 minute, 8 hours, 24 hours, and 48hours. RNA extractions were done using a NucleoSpin RNA kit(Macherey-Nagel). Sequencing was performed at BGI using anIllumina HiSeq 2000 sequencer.

Mass spectrometryGIST882 and GIST48 cells were plated in T-75 bottles, grown

until 80% confluence, and then treated with 10 mmol/L of

Table 1. PDE3A and PDE3B expression in 630 human cancerous tumors

Tumor type

Numberoftumors

PDE3Aexpressionpositive, n (%)

PDE3Bexpressionpositive, n (%)

GIST 55 50 (90) 33 (60)Other sarcomasAngiosarcoma 13 0 (0) 0 (0)Chondrosarcoma 9 0 (0) 0 (0)Fibrosarcoma 5 0 (0) 0 (0)Undifferentiated pleomorphicsarcoma

37 4 (11) 1 (3)

Leiomyosarcoma 33 12 (36) 0 (0)Liposarcoma 42 14 (33) 8 (19)Synovial sarcoma 17 2 (12) 2 (14)

Brain tumorAstrocytoma 9 0 (0) 0 (0)Glioblastoma 45 4 (9) 0 (0)Medulloblastoma 19 5 (26) 0 (0)Meningioma 8 1 (13) 1 (13)Oligodendroglioma 9 0 (0) 0 (0)Schwannoma 1 (25) 0 (0)

Breast cancerDuctal 81 0 (0) 0 (0)Lobular 3 0 (0) 0 (0)

Cholangiocarcinoma 3 0 (0) 0 (0)Colon, adenocarcinoma 6 0 (0) 0 (0)Corpus uteri, adenocarcinoma 7 1 (14) 0 (0)Hepatocellular carcinoma 22 1 (5) 0 (0)KidneyClear cell carcinoma 35 0 (0) 1 (3)Papillary cell carcinoma 18 0 (0) 2 (11)Oncocytoma 16 0 (0) 0 (0)

Lung cancerAdenocarcinoma 26 4 (15) 0 (0)Bronchioloalveolar carcinoma 22 3 (14) 0 (0)Small cell carcinoma 5 0 (0) 0 (0)

Lymphoepithelioma 2 0 (0) 0 (0)Melanoma 10 1 (10) 2 (20)Neuroblastoma 4 0 (0) 0 (0)Ovary, adenocarcinoma 16 6 (38) 1 (6)Pancreas, adenocarcinoma 8 1 (13) 0 (0)Prostate, adenocarcinoma 4 0 (0) 0 (0)Testicular cancerEmbryonal carcinoma 5 1 (20) 0 (0)Seminoma 7 0 (0) 0 (0)Teratocarcinoma 11 3 (27) 0 (0)

Urinary bladder, transitional cellcarcinoma

14 0 (0) 0 (0)

Total 630 114 (18) 51 (8)

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anagrelide hydrochloride. Cells from three replicate samples werescraped into an 8mol/L UREA at 0-, 24-, and 48-hour time points.LC-MS/MS was performed with an Orbitrap Elite hybrid massspectrometer coupled to an EASY-nLC II system (Thermo FisherScientific). LC-MS/MS analysis, MS1 quantification, and subse-quent protein identification were performed as described inelsewhere (31).

mRNA and miRNA sequencing data analysismiRNA andmRNA data were quality controlled, preprocessed,

and analyzed with SePIA (Sequence Processing, Integration, andAnalysis), which is a comprehensive transcriptomics workflow(32). Standard adapter and quality trimming were performedwith SePIA, using a FastX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) for miRNA, Trimmomatic (33) for mRNA, readalignment with Ensembl build 38, STAR (34) for mRNA, Bowtie(35) and miRBase 21 for miRNA, quantification (Cufflinks) (36)for mRNA, and HTSeqcount (37) for miRNA. Normalization anddifferential analysis of miRNA expression between GIST882and GIST48 at four times points was performed with theR package DESeq (38), using a nominal P value of 0.01 as athreshold. Log2-normalized expression was used to identifysignificant, inversely correlated miRNA and mRNA expression(P � 0.05 and Pearson correlation coefficient � �0.7). ThesemiRNA–mRNA pairs were then cross-filtered with target predic-tion databases [microT (39), Targetscan (40), microcosm (41),pita (42)] so that a pair was supported by at least one database.Fold-change values were calculated for each of these miRNAs andtarget genes using average expression values in each cell line. Genefold-change values were used as input for signal pathway impactanalysis (SPIA; ref. 43). Genes and their predicted targetingmiRNAs were then identified for each significant pathway (SPIApGFDR � 0.1).

Other assaysThe experimental procedures for IHC stainings, qPCR analyses

for PDE3A and PDE3B mRNA expression, the assays to measurecell apoptosis and cell proliferation, and Western blotting aredescribed in the Supplementary Materials and Methods.

Statistical analysisNonnormal distributions between groups were compared

with the Mann–Whitney U test. The interrater agreement inIHC scoring of PDE3A and PDE3B between two independentraters (O.-P. Pulkka and O. Tynninen) was measured bycomputing Cohen's kappa coefficient. The Wilcoxon matchedpaired test was used for tumor volume comparisons betweenthe day 1 and the end day of each in vivo experiment. TheP values are two-sided. No statistical power analyseswere done to predetermine the xenograft model samplesizes. The statistical calculations were done using the IBMSPSS Statistics package v. 22.0 (IBM) or STATISTICA 13.0(Dell Statistica).

Data availabilityThe RNA sequencing data from this study have been deposited

to the NCBI's BioSample database (https://www.ncbi.nlm.nih.gov/biosample) under the accession numbers SAMN07445399-SAMN07445421. Themass spectrometry rawdata were depositedto the Peptide Atlas (www.peptideatlas.org) under the identifierPASS01084.

ResultsPDE3 mRNA expression in human cancer

To identify potential driver oncogenes in GIST, we investigatedthe MediSapiens in silico database of human transcriptomes forgenes that are highly expressed in GISTs, and for genes withprotein products known as drug targets (44). Outlier statisticswas performed with the GTI analysis (25), which ranked the 25best mRNA expression outlier genes for GIST. This analysisidentified not only genes that are well known to be overexpressedinGIST such asKIT, anoctamin 1, and protein kinase C theta, but alsogenes such as phosphodiesterase 3A (PDE3A; Fig. 1A; Supplemen-tary Fig. S1 and Supplementary Table S1). The relative expressionsof PDE3A and PDE3B genes in different histologic types ofsarcoma are depicted in Fig. 1B.

PDE3 protein expression in human cancersExpression of PDE3A and PDE3B was next investigated in a

tissuemicroarray consisting of 36 human tumor types originatingfrom 630 individuals using IHC [Table 1; KIT and ANO1 (45)expressions in these tumors are provided in SupplementaryTable S2]. In line with the in silico outlier analysis, PDE3A wasexpressed in 50 (90.9%) of the 55 GISTs investigated, and PDE3Bin 33 (60.0%), but both were only infrequently expressed in theother tumor types examined. However, PDE3A expression wasrelatively common also in ovarian adenocarcinoma [six (37.5%)out of 16], leiomyosarcoma [12 (36.4%) out of 33], and lipo-sarcoma [14 (33.3%) out of 42]. The interrater agreement in IHCscoring of GIST PDE3A and PDE3B expressions was comparedbetween two independent raters on 150 samples, and the agree-ment turned out to be very good [for PDE3A, the kappa coefficientwas 0.85, 95% confidence interval (CI), 0.80–0.90; for PDE3B,0.82, 95% CI, 0.76–0.88].

Both PDE3A and PDE3B showed uniform staining inGIST cellsin the seven GISTs examined from whole tissue sections (Fig 1C).Neuronal cells, some smooth muscle cells, and the interstitialcells of Cajal also expressed PDE3A in histologically normalintestinal tissues (Supplementary Fig. S2), whereas PDE3B wasnot expressed in any cell type in the intestinal tissue. PDE3Aand PDE3B mRNA expression was investigated using qPCR from7 GISTs and 30 leiomyosarcomas of the tissue microarrayseries from which enough tissue was available for this analysis.The qPCR results were generally in agreement with the IHCresults (Fig. 1D) and with the transcriptome data available inMediSapiens ISTOnline database (Fig. 1B; http://ist.medisapiens.com/).

Drug sensitivity testing of GIST cell linesTwo GIST cell lines, GIST48 and GIST882, were next tested for

sensitivity to 200 drugs, and the drug–response profiles werecompared with the expression of the 25 genes that were highlyexpressed inGIST (Fig. 1A; Supplementary Table S3). As expected,several tyrosine kinase inhibitors such as imatinib, nilotinib,sunitinib, and dasatinib effectively reduced the viability of theGIST cell lines. Interestingly, anagrelide showed efficacy onGIST882 cells but not on GIST48 cells (Fig. 1E). The GIST48 cellline showed higher sensitivity to imatinib than the GIST882 cellline.

Effects of anagrelide in GIST cell linesWe next investigated the effects of four PDE3 inhibitors, ana-

grelide, amrinone, milrinone, and cilostazol, in the GIST48 and

Anagrelide for GIST




http://hannonlab.cshl.edu/fastx_toolkit/http://hannonlab.cshl.edu/fastx_toolkit/https://www.ncbi.nlm.nih.gov/biosamplehttps://www.ncbi.nlm.nih.gov/biosamplewww.peptideatlas.orghttp://ist.medisapiens.com/http://ist.medisapiens.com/http://clincancerres.aacrjournals.org/

Figure 1.

High PDE3A and PDE3B expression is frequent in GIST. A,mRNA expression profiles of GISTs were ranked using the GTI outlier statistics in the MediSapiens ISTOnline database. Genes (n¼ 25) with the highest mRNA expression in GIST as compared with the reference samples were identified. B, A box-whisker plotshowing the relative PDE3A and PDE3BmRNA expression in soft-tissue sarcoma samples available in the MediSapiens database. The number of samples studiedis indicated in the brackets. The bottom of the box is the 25th percentile of the data, the top of the box is the 75th percentile, and the horizontal line is the median.The whiskers extend to 1.5 times the interquartile range from the edges of the box, and any data points beyond this are considered outliers, marked by hollowcircles. C, Examples of lacking (left) and present (right) PDE3A and PDE3B protein expression in GIST tissue samples (magnification,�200; scale bar, 100 mm).D, PDE3A and PDE3B protein expression at IHC, and the normalized PDE3A and PDE3BmRNA expression measured with qPCR from the same tumors. E, The 20most effective drugs ranked by the DSS for GIST882 and GIST48 cell lines based on high-throughput drug sensitivity profiling with 200 anticancer compounds.

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GIST882 cell lines. As imatinib, anagrelide induced a cytotoxiceffect in the GIST882 cell line at a submicromolar concentration(IC50 ¼ 0.016 mmol/L), but it was only weakly active in theGIST48 cell line (Fig. 2A–C).Of the compounds tested, anagrelideinhibited PDE3 activity most effectively leading to an increase inthe intracellular cAMP and cGMP levels in GIST cells (Fig. 2D).

Effect of PDE3 inhibition on GIST cell viabilityThe role of PDE3 in GIST cells was next investigated by pre-

venting its expression using siRNAs. PDE3A inhibition did notinfluence cell proliferation, whereas PDE3B inhibition decreasedslightly the proliferation ofGIST882 cells, and less thanKIT siRNA(Fig. 3A).Downregulation ofKITorPDE3Bwith siRNAs increasedapoptosis in both cell lines studied in a TUNEL assay 72 hoursafter siRNA transfections (Fig. 3B). These findings suggest thatPDE3 inhibition may influence more cell apoptosis thanproliferation.

When we treated GIST882 cells with PDE3 siRNAs and increas-ing concentrations of anagrelide, both PDE3B siRNA and acombination of PDE3A siRNA and PDE3B siRNA increased thecytotoxic effect of anagrelide, whereas PDE3A siRNAhad rather anopposing effect, and none of the treatments influenced markedlyGIST48 viability (Fig. 3C; Supplementary Fig. S3A). These resultssuggest that PDE3 inhibition may have only a limited effect onGIST882 cell survival and that PDE3A is required to mediateanagrelide-associated cytotoxicity.

When the GIST882 cells were treated with 0.5 mmol/L ana-grelide and rising concentrations (from 0.0001 mmol/L to30 mmol/L) of PDE inhibitors cilostazol, milrinone, or amri-none for 72 hours, cilostazol andmilrinone abolished the effectof anagrelide in GIST882 cells, whereas amrinone had littleeffect, and none of the treatments had a substantial effect on theviability of GIST48 cells (Fig. 3D; Supplementary Fig. S3B). Thissuggests that anagrelide induces cell death via the PDE3s andthat competitive binding of other compounds to the PDEs mayabolish the effect. PDE3A or PDE3B attenuation with siRNAs oranagrelide had no effect on KIT or phospho-KIT expression(Fig. 3E). It has been suggested that SLFN12 is required forPDE3A-mediated cell death in GIST and that imatinib reducesPDE3A expression (17). We did not detect SLFN12 expressionin the GIST cell lines either before or after the anagrelidetreatment, and imatinib or siKIT treatments had no effect onPDE3 expression in the cell lines (Fig 3E and F).

Effect of anagrelide on GIST cell signalingTo identify the signaling pathways that are influenced with

anagrelide treatment, the miRNA and mRNA expression of theGIST882 and GIST48 cell lines was investigated using RNAsequencing, and protein expression was studied with label-freeLC/MS. We used a comprehensive transcriptomics data analysisand integration workflow (32) to identify those gene transcriptsfrom expression data that were inversely expressed as comparedwith their putative targeting miRNAs. Pathway impact analysis ofthe gene transcripts identified associated pathways that wereperturbed in the GIST882 cell line after 48 hours of anagrelidetreatment, such as oocyte meiosis, cell cycle, focal adhesion,neurotrophin signaling, protein processing in the endoplasmicreticulum, and insulin signaling (Supplementary Fig. S4 andSupplementary Table S4). These pathways were relatively unper-turbed in the GIST48 cell line. The genes associated with theperturbed pathways were further characterized with the corre-

sponding peptide expression to identify the genes that are affectedmost by anagrelide treatment. These genes were defined as havingpeptide expression highly correlated to the corresponding genetranscript expression and an inverse association with the targetingmiRNA (Supplementary Materials and Methods). The identified15 gene transcripts included genes such as the protein phosphatase 1subunit (PPP1CB) and the 14-3-3 family protein YWHAQ (Supple-mentary Table S5), both partners of the PDE3 signalosomes (33).

Patient-derived mouse GIST xenograft modelsWe finally investigated the in vivo efficacy of anagrelide in three

patient-derived GIST xenograft mouse models and in a GIST822cell line–derived xenograft model. Anagrelide inhibited orreduced tumor growth in three of these four models (Fig. 4). Themost potent effectwas observed in theGIST2Bmodel that harborsa KIT exon 9 mutation leading to p.A502_Y503 duplication. Inthis model, anagrelide reduced the tumor volume 68% after10 days of therapy, and was more effective than imatinib(Fig. 4B). In the GIST3 model with KIT exon 11 indel mutation(p.W557_V559delinsF), imatinib was more effective than ana-grelide, and in the GIST9 model with KIT primary exon 11mutation and a secondary exon 17 mutation, none of the treat-ments was effective. In the GIST882 xenograft model, imatiniband anagrelide stabilized the tumor growth when each was givenas a single agent, but their combination reduced the tumorvolume (Fig. 4B). When the resected tumor samples were gradedfor histologic response, the most pronounced responses wereobtained with the imatinib plus anagrelide combination inthe three models where the treatments were active (Fig. 4C;Supplementary Fig. S5). None of the treatments influencedmarkedly GIST PDE3A or PDE3B expression levels or KIT signal-ing pathway activity (Supplementary Figs. S6 and S7). GIST PDE3expression levels showed noobvious associationwith response toanagrelide, but, interestingly, in the treatment-resistant GIST9model, the PDE3A to PDE3B ratio was higher than in the threeGIST models that were sensitive to anagrelide either as a singleagent or in combination with imatinib (Supplementary Fig. S8Aand S8B). We studied whether SLFN12 is expressed in the GISTxenograft mouse models and found that SLFN12 expressionincreased in all anagrelide-treated tumor tissues, except in theunresponsive GIST9 model, as compared with a nontreatedcontrol group (Supplementary Fig. S8C).

DiscussionWe found high PDE3A and PDE3B expression in GISTs as

compared with several other human tumors or histologicallynormal tissues. Besides tyrosine kinase inhibitors, known to beeffective for GIST based on clinical trials, also anagrelide, aphosphodiesterase 3 inhibitor marketed for the treatment ofpatients with thrombocythemia, was cytotoxic on the GIST882cell line. In three of the four GIST xenograftmodels that were usedfor testing, anagrelide had antitumor activity. Taken together,these findings suggest that phosphodiesterases have a role in thepathogenesis of GIST, and that some phosphodiesterase inhibi-tors might be active agents in the treatment of GIST.

Because PDE3's expression was frequent in GISTs and rare inthe other tumor types examined, we hypothesized that PDE3activity might be important for GIST viability. In an agreementwith this hypothesis, in a GIST murine model harboring a germ-line KIT mutation (K641E), PDE3A was expressed in the gastric

Anagrelide for GIST





antrum and in the interstitial cells of Cajal (46), the proposedprogenitor cells for GIST (47). PDE3A and its mRNA are frequent-ly expressed in GISTs (17, 18).

Overexpression of PDE isoforms and impaired cAMP or cGMPgeneration occur also in various other cancers, such as leukemiaand colorectal cancer (14, 15). PDE inhibition may impair the

Figure 2.

PDE3 inhibitor anagrelide eradicates GISTcells. A, Anagrelide reduced GIST882 cell linecell viability at low concentrations, whereasthree other PDE3 inhibitors had no significanteffect at nontoxic concentrations. B,Proliferation of GIST882 and GIST48 cell linesafter imatinib (1 mmol/L), anagrelide(10 mmol/L), or imatinib plus anagrelidetreatment; � , P < 0.001 between the DMSOtreated control group and each of the drugtreated groups on day 4. C, GIST cellapoptosis in GIST882 and GIST48 cell linesafter treatment with imatinib (1 mmol/L),anagrelide (10 mmol/L), or their combination.The P values refer to the difference betweenthe DMSO group and each of the drug treatedgroups. D, Intracellular cAMP and cGMP inresponse to DMSO (reference) and to fourPDE3 inhibitors at the concentration of10 mmol/L in GIST882 and GIST48 cell lines.The P values refer to the difference betweenthe DMSO group and each of the drugtreatment groups (data representmean� SEM).

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Figure 3.

Effects of PDE3A and PDE3B inhibition on GIST cell viability. A and B, The effect of transfection with control siRNA, KIT siRNA, PDE3A siRNA, and PDE3B siRNAon GIST cell proliferation (A) and on cell death (B) in GIST882 and GIST48 cell lines. Cell proliferation of both cell lines decreases slightly with PDE3B siRNAtreatment, but the effect is not as potent as with KIT knockdown. Downregulation of PDE3B increased the proportion of TUNEL-positive (apoptotic) cells in theGIST882 cell line and tended to increase the proportion of apoptotic cells in the GIST48 cell line 48 hours after treatment with the siRNA. C, Effect of thecombinations of PDE3 siRNAs and anagrelide on the GIST882 and GIST48 cell lines. siPDE3B and siPDE3A/B reduced GIST882 cell viability already at the smallestconcentration tested (0.1 nmol/L), whereas siPDE3A had an opposite effect, and none of the treatments influencedmarkedly GIST48 viability. SEM < 0.05 in alldata points. D, Effect of the combination of PDE3 inhibitors cilostazol, milrinone, and amrinone given together with anagrelide (0.5 mmol/L) on GIST882 andGIST48 cell line viability. Larger concentrations of cilostazol and milrinone rescued the anagrelide-induced cytotoxicity in the GIST882 cell line. SE < 0.05 in alldata points. E, AWestern plot showing the expression of PDE3A, PDE3B, KIT, phosphorylated KIT, and actin (control) in the GIST48 and GIST882 cell lines.Expression after 72 hours of imatinib (1 mmol/L) and anagrelide (10 mmol/L) treatment, and 72 hours after siRNA transfections are shown. The arrow representsthe correct size of the PDE3B protein. Data represent mean� SEM. F,AWestern blot showing the SLFN12 expression in the GIST882 and GIST48 cell lines 72hours after anagrelide (10 mmol/L) treatment. � , P < 0.05 and �� , P < 0.01. The P values refer to the comparisons with the controls.

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growth of cancer cell lines (16), and the PDE3Amodulators 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) and cilostazol may reduce the viability of

GIST cells, and may have a synergistic effect when used withimatinib (17). In the current cell lines, cilostazol had little effect,and in agreement with this in one study, cilostazol did not

Figure 4.

A, GIST xenograft model characteristics,duration of the experiment, and tumorvolume change after starting the drugtreatment. Data represent mean� SEM.Anagrelide has antitumoral activity in GISTxenograft models. B, Tumor volume changesin 4 GIST xenograft models during treatmentwith imatinib, anagrelide, or the combinationof imatinib and anagrelide. C, Histologicresponse of GISTs to imatinib, anagrelide,and their combination in mouse xenograftmodels. Histologic response to imatinib,anagrelide, imatinib plus anagrelide, and tothe control (no treatment) in four mouseGIST xenograft models. GIST2B, GIST9, andGIST3 are subcutaneous patient-derivedxenograft models, and GIST882 is a mousetumor induced with GIST882 cell lineinoculation. Histologic response was gradedevaluating the proportion of the tumorconsisting either of necrosis, myxoiddegeneration, or fibrosis on H&E stainings.Grade 1, 0% to 10%; grade 2, >10%, but�50%; grade 3, >50%, but�90%; and grade4, >90%.

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replicate the cytotoxic effect of DNMDP in DNMDP-sensitivecell lines (48). Although several phosphodiesterase familiesregulate the cell cAMP content, PDE3 and PDE4 are the keyenzyme families in the hydrolysis of the cAMP phosphodiesterbonds (9). In the GIST cell lines tested, anagrelide inhibited thePDE3 activity most effectively of the compounds evaluated lead-ing to an increase in the intracellular cAMP and cGMP levels inGIST cells.

To investigate the role of PDE3 inhibition further in GIST cells,PDE3A and PDE3B were depleted in GIST cell lines using RNAinterference. Inhibition of PDE3B, but not of PDE3A, decreasedslightly the viability of GIST882 cells, suggesting that the cytotoxiceffect of anagrelidemay not be caused only by direct inhibition ofPDE3A or PDE3B enzyme activity. We studied further the effect ofthe combination of anagrelide, PDE3 siRNAs, and other PDE3inhibitors cilostazol, milrinone, and amrinone. We observed thatPDE3A knockdown decreased sensitivity to anagrelide, whereasPDE3B knockdown increased anagrelide-induced cytotoxicity.Cilostazol andmilrinone rescued anagrelide cytotoxicity, suggest-ing that these PDE3 inhibitors may compete with anagrelide forbinding to the same molecular target.

Cyclic nucleotide signaling is mediated via the formation ofcyclic nucleotide signalosomes, in which the PDE isoforms andother precisely recruited proteins form highly specialized proteincomplexes (49). Disruption and interference of the complexes ordislocation of complex partners may lead to an aberrant functionof the signalosome. For example, binding of the PDE3modulatorDNMDP to PDE3Apromotes the interaction between PDE3A andSchlafen 12 (SLFN12) that mediates the death of HeLa cells,whereas depletion of PDE3A rescues the cells from death (48).We observed that anagrelide may increase the expression ofSLFN12 in GIST xenograft mouse tissues, but SLFN12 expressionwas not required to induce the cytotoxic effect of anagrelide inGIST cells.We assessed the SLFN12 expression alsowith IHC fromformalin-fixed paraffin-embedded samples (data not presented),but the staining was unspecific, suggesting presence of SLFN12expression in various tissues and especially in leukocytes, but notin the interstitial cells of Cajal. The expressions of the proteinphosphatase 1 subunit PPP1CB and the 14-3-3 family proteinYWHAQ, both known partners of the signalosomes of PDE3s(49), were dysregulated at the gene expression level after anagre-lide treatment in the GIST cell lines. PDE3A and PDE3B reside inthe same protein complex (48, 50). The current data are com-patible with a hypothesis that anagrelide cytotoxicity requiresPDE3 and that the cytotoxic effect of anagrelide may be mediatedthrough modulation of the PDE3 signalosome complex that isdependent on both PDE3A and PDE3B.

Phosphodiesterases have previously been evaluated as poten-tial anticancer drugs in experimental models, but the studies havemainly focused on the PDE4 and PDE5 families. Phosphodies-terase inhibition may have antitumor efficacy in some hemato-logicmalignancies such as acute lymphoblastic leukemia andCLL(51, 52). PDE4 inhibition with rolipram suppressed the tumorgrowth and augmented the antitumor effects of chemotherapyand radiotherapy in a brain tumor model (53) and attenuatedproliferation and angiogenesis of lung cancer in a nude mousexenograft model (54). PDE4D inhibitors NVP-ABE171 and cilo-milast inhibited the growth of prostate cancer xenografts, and thePDE5 inhibitor sildenafil the growth of human colorectal cancerxenografts in nude mice (55, 56).

When we investigated the in vivo efficacy of PDE3 inhibitoranagrelide in GIST xenograft mouse models, anagrelide waseffective also in a GIST xenograft mouse model with KIT exon9mutation.KIT exon9mutationoccurs in about 10%of allGISTs,and almost all of these are duplications of codons 502 and 503leading to p.Ala502_Tyr503dup (57). GISTs with KIT exon 9mutations may pose a therapeutic challenge, as these GISTsrequire a high daily dose of imatinib (800 mg/day) for optimalefficacy, which dose is often associated with substantial adverseeffects (58).

We conclude that PDE3A and PDE3B are frequentlyexpressed in GISTs and that a PDE3-selective compound, ana-grelide, had antitumor activity for GIST in in vitro and in vivomodels. Data from a series of patients with chronic myelopro-liferative disease treated with both anagrelide and imatinibsuggest that their coadministration is feasible in the clinic (59).Therefore, a clinical trial evaluating the combination of ana-grelide plus imatinib in the treatment of advanced imatinib-resistant GIST appears warranted.

Disclosure of Potential Conflicts of InterestO. Kallioniemi holds ownership interest (including patents) inMediSapiens

and Sartar Therapeutics. H. Sihto and O.-P. Pulkka are employees of and holdownership interest (including patents) in Sartar Therapeutics. H. Joensuu is anemployee ofOrionPharma andNeutronTherapeutics, holds ownership interest(including patents) in Sartar Therapeutics, and is a consultant/advisory boardmember for Neutron Therapeutics. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design:O.-P. Pulkka, P. Laakkonen, O. Kallioniemi, H. Sihto,H. JoensuuDevelopment of methodology: O.-P. Pulkka, Y.K. Gebreyohannes,K. Wennerberg, M. Varjosalo, O. Kallioniemi, P. Sch€offski, H. JoensuuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): O.-P. Pulkka, Y.K. Gebreyohannes, A. Wozniak,S. Keskitalo, A. Murum€agi, E. Kulesskiy, K. Wennerberg, P. Laakkonen,O. Kallioniemi, P. Sch€offski, H. Sihto, H. JoensuuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): O.-P. Pulkka, Y.K. Gebreyohannes, A. Wozniak,J.-P. Mpindi, O. Tynninen, K. Icay, A. Cervera, A. Murum€agi, M. Laaksonen,M. Varjosalo, R. Lehtonen, S.Hautaniemi,O. Kallioniemi, P. Sch€offski,H. Sihto,H. JoensuuWriting, review, and/or revision of the manuscript: O.-P. Pulkka,Y.K. Gebreyohannes, A. Wozniak, O. Tynninen, K. Icay, A. Cervera,A. Murum€agi, M. Laaksonen, K. Wennerberg, M. Varjosalo, P. Laakkonen,S. Hautaniemi, P. Sch€offski, H. Sihto, H. JoensuuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Keskitalo, M. Varjosalo, H. JoensuuStudy supervision: M. Varjosalo, R. Lehtonen, H. Sihto, H. Joensuu

AcknowledgmentsThis studywas supported by the Academyof Finland, Center of Excellence for

Translational Cancer Biology, Cancer Society of Finland, Jane and Aatos ErkkoFoundation, HUCH Research Funds, Sigrid Juselius Foundation, Kom op tegenKanker (Stand up to Cancer), the Flemish Cancer Society (Belgium), IdaMontinFoundation, Emil Aaltonen Foundation, and Luise and Henrik KuningasFoundation.

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 March 12, 2018; revised July 23, 2018; accepted December 4, 2018;published first December 7, 2018.


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2019;25:1676-1687. Published OnlineFirst December 7, 2018.Clin Cancer Res Olli-Pekka Pulkka, Yemarshet K. Gebreyohannes, Agnieszka Wozniak, et al. Anagrelide for Gastrointestinal Stromal Tumor

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Anagrelide for Gastrointestinal Stromal Tumor · Translational Cancer Mechanisms and Therapy...

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