dopamine receptor d5 is a modulator of tumor response to ... · used: glioblastoma (gbm; gl805b),...

Translational Cancer Mechanisms and Therapy

Dopamine Receptor D5 is a Modulator of TumorResponse to Dopamine Receptor D2 AntagonismVarun V. Prabhu1, Neel S. Madhukar2, Coryandar Gilvary2, C. Leah B. Kline3,Sophie Oster3,Wafik S. El-Deiry3, Olivier Elemento2, Faye Doherty4,Alexander VanEngelenburg4, Jessica Durrant4, Rohinton S. Tarapore1,Sean Deacon5, Neil Charter5, Jinkyu Jung6, Deric M. Park7, Mark R. Gilbert6,Jessica Rusert8, Robert Wechsler-Reya8, Isabel Arrillaga-Romany9,Tracy T. Batchelor9, Patrick Y.Wen10,Wolfgang Oster1, and Joshua E. Allen1

Abstract

Purpose: Dopamine receptor D2 (DRD2) is a G protein–coupled receptor antagonized by ONC201, an anticancersmall molecule in clinical trials for high-grade gliomas andother malignancies. DRD5 is a dopamine receptor familymember that opposes DRD2 signaling. We investigated theexpression of these dopamine receptors in cancer and theirinfluence on tumor cell sensitivity to ONC201.

Experimental Design: The Cancer Genome Atlas was usedto determine DRD2/DRD5 expression broadly across humancancers. Cell viability assays were performed with ONC201 in>1,000 Genomic of Drug Sensitivity in Cancer and NCI60 celllines. IHC staining of DRD2/DRD5 was performed on tissuemicroarrays and archival tumor tissues of glioblastomapatients treated with ONC201. Whole exome sequencing wasperformed in RKO cells with and without acquired ONC201resistance. Wild-type and mutant DRD5 constructs were gen-erated for overexpression studies.

Results: DRD2 overexpression broadly occurs acrosstumor types and is associated with a poor prognosis.Whole exome sequencing of cancer cells with acquiredresistance to ONC201 revealed a de novo Q366R mutationin the DRD5 gene. Expression of Q366R DRD5 wassufficient to induce tumor cell apoptosis, consistentwith a gain-of-function. DRD5 overexpression in glio-blastoma cells enhanced DRD2/DRD5 heterodimersand DRD5 expression was inversely correlated withinnate tumor cell sensitivity to ONC201. Investigation ofarchival tumor samples from patients with recurrent glio-blastoma treated with ONC201 revealed that low DRD5expression was associated with relatively superior clinicaloutcomes.

Conclusions: These results implicate DRD5 as a negativeregulator of DRD2 signaling and tumor sensitivity toONC201DRD2 antagonism.

IntroductionG protein–coupled receptors (GPCR) are the largest superfam-

ily of membrane receptors in humans. However, these receptorsare underexploited therapeutic targets for oncology that controlseveral signaling pathways that are critical for cancer, includingthe integrated stress response and Ras signaling (1). Overexpres-sion of the GPCR dopamine receptor D2 (DRD2) in cancer andanticancer effects of DRD2 antagonism via induction of the

integrated stress response and inhibition of Akt/ERK signalinghave been reported in a range of tumor types (2–5).

The imipridone class of anticancer compounds share a uniquetri-heterocyclic core chemical structure (6) and selectively targetGPCRs (7, 8). ONC201 is the first imipridone to enter clinicaltrials (9) and is a selective antagonist of DRD2 and DRD3. Thiscompound has exhibited encouraging safety, pharmacokinetic,pharmacodynamics, and efficacy profiles in phase I/II trials,including sustained tumor regressions in advanced chemoresis-tant cancers such as glioblastoma, endometrial cancer, andmantlecell lymphoma (7, 10–12).DRD2antagonismbyONC201 resultsin activation of the integrated stress response (13, 14) andinactivation of Akt/ERK signaling that induces downstreamDR5/TRAIL-mediated apoptosis in cancer cells and impairs cancerstem cell self-renewal (9, 15, 16).

We investigated the dysregulation of DRD2 in human cancerand its role in tumor response to ONC201 to uncover predictivebiomarkers.

Materials and MethodsCell culture and reagents

ONC201-resistant RKO human colon cancer cells have beengenerated previously using increasing concentration gradientincubations (13). All other cell lines were obtained from the

1Oncoceutics, Philadelphia, Pennsylvania. 2Weill Cornell Medical College, NewYork, New York. 3Fox Chase Cancer Center, Philadelphia, Pennsylvania. 4His-toTox Labs, Inc., Boulder, Colorado. 5Eurofins DiscoverX Corporation, Fremont,California. 6Neuro-OncologyBranch,National Cancer Institute, National Instituteof Health, Bethesda, Maryland. 7The University of Chicago, Chicago, Illinois.8Sanford Burnham-Prebys Medical Discovery Institute, La Jolla, California.9Massachusettes General Hospital, Boston, Massachusetts. 10Dana Farber Can-cer Institute, Boston, Massachusetts.

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

Corresponding Author: Joshua E. Allen, Oncoceutics Inc., Philadelphia, PA19104. Phone: 1-844-662-6797; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-2572

�2018 American Association for Cancer Research.

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Published OnlineFirst December 17, 2018; DOI: 10.1158/1078-0432.CCR-18-2572

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ATCC and cultured as per ATCC recommendations. Cells wereauthenticated every month by bioluminescence, growth, andmorphologic observation. ONC201 was provided by Oncoceu-tics, Inc.

GPCR profilingExperimental GPCR profiling was performed utilizing the

PathHunter b-arrestin enzyme fragment complementation (EFC)assay at DiscoverX as described previously (17, 18). PathHuntercells were seeded in a total volume of 20 mL into white walled,384-well microplates, and incubated at 37�C for the appropriatetime prior to testing. For agonist determination, cells were incu-bated with test compound to induce response. Five microliters of5� test compound was added to cells and incubated at 37�C orroom temperature for 90 or 180 minutes. For antagonist deter-mination, cells were preincubated with test compound followedby agonist challenge at the EC80 concentration (5 mL of 5� samplewas added to cells and incubated at 37�Cor room temperature for30minutes and 5 mL of 6� EC80 agonist in assay buffer was addedto the cells and incubated at 37�C or room temperature for 90 or180 minutes). Final assay vehicle concentration was 1%. Assaysignal was generated through a single addition of 12.5 or 15 mL(50%, v/v) of PathHunter Detection reagent cocktail, followed by1-hour incubation at room temperature. Microplates were readfollowing signal generation with a PerkinElmer EnvisionTMinstrument for chemiluminescent signal detection. Compoundactivity was analyzed using CBIS data analysis suite (Chem-Innovation). GPCR panel was assayed with 10 mmol/L testcompound.

Genomics of drug sensitivity in cancer cell line screeningCell viability assays were performed as previously described

(19, 20) with >1,000 human cancer cell lines at 72-hourpost-ONC201 treatment to generate dose–responses curvesat concentrations from 78 nmol/L to 20 mmol/L. Cell viabilitywas determined using either a DNA dye (Syto60) or metabolicassay (Resazurin or CellTitre-Glo). Fluorescence intensitydata from screening plates for each dose–response curve arefitted using a multilevel fixed effect model (21) to generate IC50and AUC.

Propidium iodide staining and cell viability assaysCells were treated, trypsinized, ethanol-fixed, stained with

propidium iodide (Sigma), and analyzed by flow cytometry aspreviously described (9). Cell viability was determined asdescribed previously using the CellTitre-Glo reagent (Promega;ref. 15).

Western blot analysisWestern blotting was performed as described previously (8, 11,

18). Briefly, lysates were prepared and evaluated with proteinassay (Bio-Rad). LDS sample buffer and reducing agent (Invitro-gen) were added for SDS-PAGE. After transfer, primary andsecondary antibody incubations were performed, and signal wasdetected using a Chemiluminescent Detection Kit, followed byautoradiography.

ImmunohistochemistryIHC assessment of DRD2 (sc-5303, 1:300) and DRD5

(HPA048930, 1:100) expression in paraffin-embedded forma-lin-fixed archival tumor tissue of patients on trial was performedusing the automated Leica Bond Rx system followed by dehydra-tion andmounting. FollowingUSBiomax tissuemicroarrayswereused: glioblastoma (GBM; GL805B), neuroblastoma (MC602),pheochromocytoma (AD2081), medulloblastoma (BC17012c),and endometrial cancer (EMC961). Additionally, the BioChainFDA Standard Tissue Array (T8234701-1) was also used. Tissuemicroarray and patient archival tumor tissue slides stained forDRD2 and DRD5 were scanned (Aperio AT2 slide scanner) andloadedintoimageanalysis software(VisiopharmVIS).Wholeslideimageswere annotated by a pathologist to create region of interest(ROI) delineating tumor area and to set thresholds specific forDRD2- and DRD5-positive immunolabeling. Individual algo-rithms were developed to detect and stratify DRD2 or DRD5immunolabeling area for eachTMAcoreor patient sample, respec-tively. Each algorithm identified the number of pixels stainedpositively for DRD2 or DRD5 within the tumor ROI. Positively-stained pixels converted to area was quantified as a percentage oftotal tumor area. Following quantification, each sample wasreviewed to confirm appropriate algorithm application and quan-tification. The pathologist assigned a low threshold to account forbackground signal and determine a baseline positive.

Culture of medulloblastoma cells from patient-derivedxenograft

NOD-SCID IL2R-gamma (NSG) null mice used for intracranialtumor transplantation were purchased from the Jackson Labora-tory. Mice were maintained in the animal facilities at the Univer-sity of California San Diego (UCSD). All experiments were per-formed in accordance with national guidelines and regulations,and with the approval of the animal care and use committees atUCSD. Medulloblastoma PDX Med211-FH tumor cells, generat-ed in the lab of Dr. JamesM.Olson at the FredHutchinsonCancerResearch Center and Seattle Children's Hospital, were thawed andorthotopically transplanted into the cerebellums of an NSGmice.About 6 to 8 weeks later once the mice showed signs of tumorburden, they were sacrificed and the tumors were removed.Tumors were then dissociated and resuspended in NeuroCultmedium with proliferation supplement (STEMCELL Technolo-gies) and plated at 10,000 cells/well in 25 mL in a 384-well platefor ONC201 treatment. Viable cell number in each well wasdetermined using the CellTiter-Glo reagent (Promega) and read

Translational Relevance

Overexpression of dopamine receptor D2 (DRD2) is asso-ciated with a poor clinical prognosis in patients with cancer.We demonstrate that DRD5, a dopamine receptor familymember that opposes DRD2 signaling, is a negative regulatorof tumor cell sensitivity to DRD2 antagonism. Small moleculeONC201 is the first selective antagonist of D2-like dopaminereceptors for clinical oncology. We report a DRD2þDRD5�biomarker signature that is predictive of enhanced tumor cellsensitivity to ONC201 in preclinical models and is associatedwith improved outcomes in patients treated with ONC201in a phase II clinical trial for recurrent glioblastoma(NCT02525692). The predictive biomarker signature forONC201 is under evaluation in ongoing glioma clinical trialsand may be used to identify additional indications forONC201, such pheochromocytomawhich is nowbeing inves-tigated in a phase II clinical trial (NCT03034200).

Prabhu et al.

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in an automated Envision plate reader (PerkinElmer) after 48-hour incubation.

Clinical and pathologic characteristics of patients treated withONC201

We previously reported clinical outcomes and intratumoralDRD2 expression in a cohort of 17 patients with recurrent GBM(nine male and eight female) who were treated with ONC201(11). Intratumoral expression of DRD5 was determined usingarchival tumor tissue as described above and correlated withclinical outcomes. The median age of patients recruited was 57years (range 22–74 years) with WHO (2007) Grade IV histolog-ically confirmed diagnosis of GBM and median KPS of 90 (range70–100). Patients were bevacizumab na€�ve, did not have knownIDH1/2 tumor mutations, and were previously treated with atleast temozolomide and radiotherapy. MGMT status of patientswere: 2 methylated, 13 unmethylated, and 2 unknown.

Computational analysesFor the Cancer Genome Atlas (TCGA) analyses, the individual

survival scores of each included cancer type were scaled to a 0 to 1scale according to the following formula: [Value–Min(Values forCancer Type))/(Max(Values for Cancer Type)–Min(Values forCancer Type)]. For each cancer type, a z-score was calculatedbased on the log10(expression þ 1). A Fisher exact test was usedtomeasure significancewith an expression z-score cutoff of 1 andanormalized survival value cutoff of 0.5. All datawere downloadedfrom TCGA. For the GBM cohort analysis, we performed alikelihood ratio chi-square two-tailed test to examine the signif-icance of these results at 455 days of follow-up.

The generalized linear model was performed on the genomicsof drug sensitivity in cancer (GDSC) efficacy testing data with thecaret package and the R statistical programming language. Expres-sion values of DRD1 to DRD5 in all cells were used to predict theoverall efficacy of ONC201 in that same cell line (measured byIC50). The normalized coefficient score was obtained by dividingthe coefficient of each individual variable (DRD1–DRD5) by themaximum of the absolute value of all 5 coefficients: Coefn/Max(Abs(CoefDRD1-DRD5)).

The loss-of-function (LoF) essentiality data were downloadedvia Project Achilles. The shRNA LoF screening results were col-lected from Version 2.20.2 and precalculated DEMETER valueswere used as essentiality scores for DRD2. Drug efficacy scores forONC201 were collected from the GDSC cell line sensitivityscreening panel. The correlation between drug efficacy, AUCscores, and DRD2 DEMETER essentiality scores for brain cancercell lines were evaluated using the Spearman Correlation test.

ResultsDRD2 is overexpressed in human cancer

Investigation of RNA sequencing (RNA-seq) data in TCGArevealed that DRD2 is expressed broadly among human cancers,however somatic mutations are infrequent (Supplementary Fig.S1A and S1B). Comparing DRD2 expression in malignant versuscorresponding normal tissues revealed selective overexpression ofthe receptor in numerous tumor types (Fig. 1A). Poolingmultipletumor types in TCGA that are annotated with survival outcomes,we observed that patients with high levels of DRD2 expressionhad relatively inferior overall survival (Fig. 1B). IHC analysis oftissue microarrays corroborated TCGA observations that malig-nant DRD2 expression was highest in pheochromocytoma/para-

ganglioma (PCPG), GBM, neuroblastoma, medulloblastoma,and endometrial cancer (Fig. 1C and D; Supplementary Fig.S2A–S2D), which are tumor types that are sensitive to ONC201in vitro (Supplementary Fig. S3A).

DRD2 dysregulation in high-grade gliomas and concordancewith tumor response to ONC201

DRD2 exhibited significant essentiality scoring derived fromlarge-scale CRISPR screens (22) across numerous cancer cell linesassociated with various primary sites of disease (Fig. 2A). Centralnervous system cancers had the highest DRD2 gene essentialityscores, indicating that these cancer types are the most vulnerableto DRD2 antagonism among the panel. Given these observationsand the activity of ONC201 in high-grade glioma preclinicalmodels and patients (9, 11, 23), we further investigated DRD2expression in this tumor type. DRD2was highly expressed relativeto the other four dopamine receptors (Supplementary Fig. S1C),however genetic aberrations were rare among the 273 evaluatedGBM specimens: gene amplification in three specimens (1.1%),R219Hmutation in one specimen (0.4%), and no gene copy loss(Supplementary Fig. S1D). Patients with high DRD2 expressiontended to have primary, rather than secondary, GBM (Fig. 2B).Patients who had relatively long overall survival (>2 years)exhibited low expression of DRD2 whereas patients with inferiorsurvival had heterogeneousDRD2 expression, suggesting that lowDRD2 expression is necessary, but not sufficient, for relativelylong overall survival (Fig. 2C). A similar trend was observed inlow-grade glioma with disease-free survival (SupplementaryFig. S1E).

A correlation between DRD2 mRNA and ONC201 GI50 wasobserved among a panel of six GBM cell lines in the NCI60 panel(Fig. 2D), but not other tumor types (Supplementary Fig. S3B).Similarly, we found a significant concordance (cor¼ 0.57, P-value< 0.05) between a cell line's sensitivity to ONC201 within theGDSC panel and cell line-specific DRD2 gene essentiality scoresfor brain cancers (Fig. 2E). Additionally, we observed a modestconcordance between tumor type sensitivity to ONC201 in theGDCS panel and tumor type sensitivity to DRD2 RNAi by ATARiSscoring (Supplementary Fig. S3C). Lymphoma, a tumor thatONC201 is being evaluated in a phase I/II trial (NCT02420795;ref. 7), exhibited the strongest sensitivity to both DRD2 RNAi byATARiS scoring and ONC201 response by cell viability.

DRD5 is an inversely correlated predictive biomarker ofONC201 efficacy

To further evaluate the relative contribution of dopaminereceptors (DRD1–DRD5) to the efficacy of ONC201 among theGDSC panel, we used a generalized linear model to combinethe expression of all five receptors into a single model. We thenranked the relative contribution of each dopamine receptorbased on the coefficient value assigned to it by the generalizedlinear model (Supplementary Fig. S3D). In accordance with ourprevious results, we found that the strongest negative contrib-utor was DRD2—where a negative contribution denotes adecreased IC50 value as expression increases. Interestingly, wefound that DRD5 had the highest positive score—indicatingthat low expression of DRD5 was correlated with enhancedsensitivity to ONC201. DRD5 is a Gs-coupled D1-like dopa-mine receptor that exhibits downstream signaling effects (e.g.,cAMP production) that counteract D2-like receptors that areGi-coupled, such as DRD2.

DRD5 Modulates Tumor Response to DRD2 Antagonism

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To further explore biomarkers of tumor response to ONC201,we performed whole exome sequencing of RKO cell line cloneswith and without acquired resistance to ONC201 that wepreviously generated (13). Analysis of nonsynonymous muta-tions revealed a consensus heterozygous Q366R mutation inthe DRD5 gene that was exclusive to the resistant clones(Fig. 3A and B). Given that the sequenced cells were drivenby complete and stable resistance to ONC201, we hypothesizedthat the acquired resistance clones evolved to be DRD2-independent due to stable inactivation of DRD2 signaling viaDRD5. In accordance with Q366R acting as a mutation inDRD5 that enhances its ability to antagonize DRD2 signaling,Q366R DRD5 overexpression transiently induced tumor celldeath to a greater extent than the wild-type (WT) gene (Fig. 3C).Dimerization of DRD2/DRD5 can occur in cells via electrostaticinteractions between intracellular residues (24). Overexpres-sion of WT or Q366R DRD5 was sufficient to induce DRD2oligomers that are presumed to be DRD2/DRD5 heterodimers(Fig. 3D). In accordance with the hypothesis that ONC201sensitivity is associated with innately low DRD5 expression tofacilitate DRD2 downstream signaling, overexpression of WTDRD5 prior to treatment conferred resistance to ONC201-mediated apoptosis (Fig. 3E). These results suggest that DRD5is a direct negative regulator of DRD2 signaling that can

influence innate tumor cell sensitivity to DRD2 antagonismby ONC201.

Interrogation of TCGA and tissue microarrays revealed hetero-geneous malignant and normal tissue DRD5 expression that wasgenerally lower in magnitude than DRD2 and exhibited a differ-ent spectrum of expression across tumor types (Fig. 3F; Supple-mentary Fig. S2E). Somatic alterations were more common in theDRD5 gene compared with DRD2, in particular missense muta-tions, however the Q366Rmutation was not found (Supplemen-tary Fig. S3E). These results indicate that the mechanisms ofdysregulation in oncology may be distinct for these two dopa-mine receptors. A similar analysis of the clinical prognostic impactof DRD5 expression and clinical outcome did not yield a signif-icant relationship (Supplementary Fig. S3F), unlike DRD2, sug-gesting that the role of DRD5 in cancer may be limited toinfluencing response to DRD2 antagonism.

Exploring the influence of DRD5 on innate tumor cell sensi-tivity, we found that DRD5 mRNA levels are inversely correlatedwith ONC201 sensitivity in the NCI60 panel (Fig. 3G; Supple-mentary Fig. S3G and S3H). The hypothesis that DRD2þDRD5�tumor cells are highly responsive to ONC201 held true in theGDSC panel (Fig. 3H). Thus, DRD5 is a negative regulator ofDRD2 signaling that is associated with decreased tumor cellsensitivity to DRD2 antagonism by ONC201.

Figure 1.

DRD2 is selectively overexpressed in human cancer. A, Expression of DRD2 in human tumors and normal tissue in TCGA. B, z-score of expression of DRD2 mRNAlog(expressionþ 1) versus normalized survival values for different cancers in TCGA (P¼ 0.0348, Fisher exact test). C,Quantification and (D) exemplary imagesfrom DRD2 IHC analysis of tissue microarrays in GBM (n¼ 35), neuroblastoma (n¼ 25), medulloblastoma (n¼ 20), endometrial cancer (n¼ 32), andpheochromocytoma (n¼ 30) with corresponding normal tissue. Dotted lines represent average expression for each tumor type. Quantification for normal tissuecould not be performed due to low number of samples. Data are presented as DRD2-positive staining as a percentage of total tumor area.

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Given the influence of DRD5 on tumor cell sensitivity to DRD2antagonism, we evaluated the impact of dopamine receptorselectivity on the efficacy of DRD2 antagonism in cancer cells.We found that DRD2-specific antagonism results in anticancereffects in vitro that are superior to concomitant antagonism ofDRD2 and DRD5 (Supplementary Fig. S4A and S4B). AlthoughONC201 has been previously described to be highly selective forDRD2/DRD3 (25), antipsychotics such as haloperidol and chlor-promazine affect several dopamine receptors and other GPCRs(Supplementary Table S1; Supplementary Fig. S4C and S4D) atconcentrations required to kill cancer cells (2, 5). Accordingly,ONC201 demonstrated a wide and superior therapeutic windowin vitro (Supplementary Table S2).

Intratumoral DRD5 expression is associated with clinicaloutcomes in patients with ONC201-treated recurrentglioblastoma

We previously reported that DRD2 was expressed in archivaltumor specimens of patients with recurrent GBM who weretreated with ONC201 (11). Based on the observation that DRD5is a predictive biomarker for ONC201 in vitro, we evaluated the

expression of DRD5 in these archival tumor specimens by IHCanalysis. In support of DRD5-low tumors being more responsiveto ONC201, the three patients with PFS > 5 month, used as asurrogate endpoint for clinical benefit in this patient population(26), uniformly had no detectable expression of DRD5 unlikethose with PFS

Figure 3.

DRD5 is an inversely correlated predictive biomarker of tumor cell sensitivity to ONC201. A, DRD5 missense mutation identified by whole exomesequencing in RKO cells with acquired resistance to ONC201. Points are colored based on the type of genomic event. Overlapping mutation in DRD5among resistant clones is annotated with an arrow. B, Snake plot of Q366 amino acid location in DRD5 protein. C, Propidium iodide staining and (D)Western blot analysis of RKO cancer cells following overexpression of WT or Q366R DRD5 constructs. Quantitation of DRD2 monomer and dimerexpression normalized to GAPDH is shown on the right. E, Western blot analysis of PARP cleavage in HCT116 cancer cells treated with ONC201 withoverexpression of WT or Q366R DRD5. Quantitation of cleaved PARP normalized to total PARP and Ran is shown at the bottom. F, Expression ofDRD5 in human tumor samples in TCGA. G, ONC201 GI50 of NCI60 cells categorized by DRD5 mRNA expression using z-score (48 hours). H, ONC201efficacy distributions based on DRD2 and DRD5 expression in the GDSC panel (P = 0.0037 for DRD2þ/DRD5� versus DRD2�/DRD5�, KS test,72 hours). Cutoffs for DRD2 expression were z-scores of 1 and �1. Cutoffs for DRD5 were z-scores of 0.5 and �0.5.

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ongoing phase II clinical trial with ONC201 (NCT03034200).Leiomyosarcoma was also found to exhibit this presumablyfavorable expression pattern (Supplementary Fig. S5C). Leiomyo-sarcoma is a soft tissue sarcoma that oftenmanifests in the uterus,which is interesting given that ONC201 has exhibited signs ofclinical efficacy in endometrial cancer that is another type ofuterine cancer being evaluated in clinical trials (NCT03099499,NCT03485729, NCT03394027).

DiscussionGPCRs are targeted by 30% to 50% of marketed drugs and are

dysregulated inmany types of human cancer, but have historicallynot been targeted in oncology outside of select neuroendocrinetumors (1). Several studies support the notion that dopamine andother neurotransmitters play a key role in tumorigenesis andcould serve as therapeutic targets (27). Meta-analyses on cancer

Figure 4.

Intratumoral DRD5 expression is associated with clinical outcomes of patients with ONC201-treated adult recurrent glioblastoma. A, DRD5 expression by IHCanalysis in archival tumor samples categorized by PFS > 5 months (n¼ 3) or PFS < 5months (n¼ 12) of ONC201-treated recurrent GBM patients (625 mg every 3weeks orally). Data are presented as DRD5-positive staining as a percentage of total tumor area. B, Exemplary DRD5 expression by IHC analysis in archival tumorspecimens from patients with ONC201-treated recurrent GBMwho experience an objective response or progressive disease. C,Overall survival of patients withONC201-treated recurrent GBMwho had archival tumor specimens that were DRD5� versus DRD5þ by IHC analysis. Gray circles indicate a censoring event forfollowup. D, Thalamic and parietal lobe GBM lesions before and 17 months after 625 mg every 3 weeks ONC201. E,Overall tumor burden over time in respondingpatient with recurrent GBM treated with ONC201.






incidence in patients with Parkinson's disease and schizophreniapopulations have found that dopamine pathway blockade isassociated with lower levels of cancer (28, 29). Our findingsdemonstrate that DRD2 is overexpressed in a number of tumortypes and correlated with poor survival outcomes. In particular,DRD2 expression is dysregulated in gliomas that appear to be thetumor type that is most susceptible to DRD2 antagonism. Thesefindings are consistent with other studies that have demonstratedDRD2 overexpression and the anticancer effects of DRD2 antag-onism in various tumor types (2–4).

Dopamine receptors are classified as D1-like (DRD1 andDRD5) that associate with the Gs to activate adenylyl cyclase andD2-like (DRD2, DRD3, and DRD4 receptors) that couple with Gito inhibit adenylyl cyclase activity (30). Amachine learning-basedtarget identification platform (25) previously predicted thatONC201 directly antagonizes DRD2 and DRD3. b-Arrestin andcAMP assays confirmed that ONC201 selectively antagonizesDRD2/3. Schild analyses and radioligand competition assaysrevealed DRD2 antagonism at concentrations consistent withONC201 anticancer activity (25). Induction of serum prolactin,a surrogate biomarker of DRD2 antagonism, was detected inONC201-treated patients (10, 11). Additionally, disruptingDRD2 expression in tumor cells modulated ONC201-mediatedapoptosis and induction of integrated stress response (31).

In this study, we observed a correlation between DRD2 mRNAexpression and ONC201 anticancer efficacy in select tumor types.We also found concordance between tumor type sensitivity toONC201 and tumor type sensitivity toDRD2genetic knockdown.Importantly, we found that selective targeting of DRD2 wassuperior to concurrent targeting of D1-like dopamine receptors.These suggest that the selectivity of ONC201, in addition toenabling its safety profile, may be critical for optimal anticanceractivity that is more pronounced than many antipsychotics thatantagonize dopamine receptors nonspecifically.

Although prior studies have evaluated roles for DRD2 (2–4)andDRD4 (32) in cancer, the role ofDRD5 in cancer has not beenwell studied. One study showed that a DRD5 agonist suppressespituitary, glioblastoma, colon, and gastric tumor growth (33),whereas another revealed DRD5 upregulation in response todocetaxel in non–small cell lung cancer (34). The results that wereport implicate DRD5 as a negative regulator of DRD2 antago-nism with utility that may be considered in distinct contexts. Thefirst consideration is that innate tumor cell expression of DRD5 isinversely associated withDRD2-associated downstream signalingeffects and therefore response to DRD2 antagonism. Accordingly,innate DRD5 expression was inversely correlated with ONC201activity in vitro and in patients. A second consideration is thattransient DRD5 activation in cancer cells leads to decreasedDRD2-associated downstream signaling that will produce ananticancer response. This is consistent with the observation thattumor cells with stable acquired resistance to ONC201 possesseda Q366R mutation in the DRD5 gene that induced apoptosisupon expression in parental cells. These two distinct considera-tionsmay lead to theuse ofDRD5as a predictive biomarker and asa therapeutic target in conjunction with DRD2 antagonism,respectively. Further to the former utility, a DRD2þDRD5�expression signature pointed to PCPG and uterine cancer aspotential indications for ONC201 clinical studies. The expressionsignature could provide additional clinical opportunities toexpand the utility of ONC201 in other gliomas with low DRD5expression and guide the selection of other DRD5-low tumors

beyond glioma for further evaluation with ONC201. Addition-ally, this predictive biomarker could help identify combinatorialtherapies that can lower innate DRD5 expression and sensitizetumor cells to ONC201.

One limitation of this study is that although DRD2 is clearlyoverexpressed in cancer, the mechanism of DRD2 overexpressionthat can be detected at the mRNA level needs further study. Therole of DRD2 and DRD5 homodimers versus heterodimers intumor growth, spectrum of expression, and response to ONC201should also explored. Finally, the use of intratumoral DRD5expression to predict clinical outcomes in patients withONC201-treated high-grade glioma needs to be validated pro-spectively in a larger number of patients and in other tumor types.

Together, our results posit DRD2 as a therapeutic target foroncology that can be selectively addressed by ONC201 andidentify DRD5 as a direct negative regulator of DRD2 signalingand tumor cell response to its antagonism.

Disclosure of Potential Conflicts of InterestR.S. Tarapore reports ownership interest inOncoceutics. V.V. Prabhu holds

ownership interest (including patents) in Oncoceutics. W.S. El-Deiry holdsownership interest (including patents) in and is a consultant/advisory boardmember for Oncoceutics. I. Arrillaga-Romany reports receiving speakersbureau honoraria from Merck and is a consultant/advisory board memberfor Insys, Karus, Agios, and Boehringer Ingelheim. T.T. Batchelor reportsreceiving commercial research grants from Pfizer and is a consultant/advisoryboard member for Merck, NXDC, Amgen, Roche, Oxigene, FoundationMedicine, Proximagen, Genomicare, UpToDate, Champions Biotechnology,Research to Practice, Oakstone, Imedex, and Jiahui Health. W. Oster holdsownership interest (including patents) in Oncoceutics. J.E. Allen reportsreceiving commercial research grants from and holds ownership interest(including patents) in Oncoceutics. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: V.V. Prabhu, N.S. Madhukar, S. Oster, D.M. Park, R.Wechsler-Reya, I. Arrillaga-Romany, W. Oster, J.E. AllenDevelopment of methodology: V.V. Prabhu, S. Oster, F. Doherty, A. VanEnge-lenburg, I. Arrillaga-Romany, J.E. AllenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V.V. Prabhu, C.L.B. Kline, W.S. El-Deiry, A. VanEnge-lenburg, J. Durrant, R.S. Tarapore, S. Deacon, N. Charter, D.M. Park, J. Rusert,I. Arrillaga-Romany, T.T. BatchelorAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):V.V. Prabhu,N.S.Madhukar,C.Gilvary,O. Elemento,R.S. Tarapore, D.M. Park, R. Wechsler-Reya, P.Y. Wen, J.E. AllenWriting, review, and/or revision of the manuscript: V.V. Prabhu, N.S.Madhukar, W.S. El-Deiry, J. Durrant, D.M. Park, M.R. Gilbert, R. Wechsler-Reya,I. Arrillaga-Romany, T.T. Batchelor, P.Y. Wen, W. Oster, J.E. AllenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V.V. Prabhu, S. Oster, W.S. El-Deiry, J. Durrant,M.R. Gilbert, I. Arrillaga-Romany, T.T. Batchelor, J.E. AllenStudy supervision: I. Arrillaga-Romany, J.E. AllenOther (experiment and generation of biological data): J. Jung

AcknowledgmentsWe thank Joseph Rucker and BenjaminDoranz for technical advisement and

assistance with DRD2 and DRD5 studies. This work was supported by grantsfrom theMusella Foundation to J.E. Allen and theNIH (CA192427) toW.Oster.

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 August 8, 2018; revised October 17, 2018; accepted December 10,2018; published first December 17, 2018.

Prabhu et al.





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2019;25:2305-2313. Published OnlineFirst December 17, 2018.Clin Cancer Res Varun V. Prabhu, Neel S. Madhukar, Coryandar Gilvary, et al. Dopamine Receptor D2 AntagonismDopamine Receptor D5 is a Modulator of Tumor Response to

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dopamine receptor d5 is a modulator of tumor response to ... · used: glioblastoma (gbm; gl805b),...

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