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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.
ClinicalCancerResearch
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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).
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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
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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.
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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.
Clin Cancer Res; 25(7) April 1, 2019 Clinical Cancer
Research2312
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al. Dopamine Receptor D2 AntagonismDopamine Receptor D5 is a
Modulator of Tumor Response to
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