Dopamine Receptor D5 Is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism Varun V

Published OnlineFirst December 17, 2018; DOI: 10.1158/1078-0432.CCR-18-2572 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism Varun 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– Results: DRD2 overexpression broadly occurs across coupled receptor antagonized by ONC201, an anticancer tumor types and is associated with a poor prognosis. small molecule in clinical trials for high-grade gliomas and Whole exome sequencing of cancer cells with acquired other malignancies. DRD5 is a dopamine receptor family resistance to ONC201 revealed a de novo Q366R mutation member that opposes DRD2 signaling. We investigated the in the DRD5 gene. Expression of Q366R DRD5 was expression of these dopamine receptors in cancer and their sufficient to induce tumor cell apoptosis, consistent influence on tumor cell sensitivity to ONC201. with a gain-of-function. DRD5 overexpression in glio- Experimental Design: The Cancer Genome Atlas was used blastoma cells enhanced DRD2/DRD5 heterodimers to determine DRD2/DRD5 expression broadly across human and DRD5 expression was inversely correlated with cancers. Cell viability assays were performed with ONC201 in innate tumor cell sensitivity to ONC201. Investigation of >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell archival tumor samples from patients with recurrent glio- lines. IHC staining of DRD2/DRD5 was performed on tissue blastoma treated with ONC201 revealed that low DRD5 microarrays and archival tumor tissues of glioblastoma expression was associated with relatively superior clinical patients treated with ONC201. Whole exome sequencing was outcomes. performed in RKO cells with and without acquired ONC201 Conclusions: These results implicate DRD5 as a negative resistance. Wild-type and mutant DRD5 constructs were gen- regulator of DRD2 signaling and tumor sensitivity to ONC201 erated for overexpression studies. DRD2 antagonism. Introduction integrated stress response and inhibition of Akt/ERK signaling have been reported in a range of tumor types (2–5). G protein–coupled receptors (GPCR) are the largest superfam- The imipridone class of anticancer compounds share a unique ily of membrane receptors in humans. However, these receptors tri-heterocyclic core chemical structure (6) and selectively target are underexploited therapeutic targets for oncology that control GPCRs (7, 8). ONC201 is the first imipridone to enter clinical several signaling pathways that are critical for cancer, including trials (9) and is a selective antagonist of DRD2 and DRD3. This the integrated stress response and Ras signaling (1). Overexpres- compound has exhibited encouraging safety, pharmacokinetic, sion of the GPCR dopamine receptor D2 (DRD2) in cancer and pharmacodynamics, and efficacy profiles in phase I/II trials, anticancer effects of DRD2 antagonism via induction of the including sustained tumor regressions in advanced chemoresis- tant cancers such as glioblastoma, endometrial cancer, and mantle cell lymphoma (7, 10–12). DRD2 antagonism by ONC201 results 1Oncoceutics, Philadelphia, Pennsylvania. 2Weill Cornell Medical College, New in activation of the integrated stress response (13, 14) and York, New York. 3Fox Chase Cancer Center, Philadelphia, Pennsylvania. 4His- inactivation of Akt/ERK signaling that induces downstream toTox Labs, Inc., Boulder, Colorado. 5Eurofins DiscoverX Corporation, Fremont, DR5/TRAIL-mediated apoptosis in cancer cells and impairs cancer California. 6Neuro-Oncology Branch, National Cancer Institute, National Institute stem cell self-renewal (9, 15, 16). 7 of Health, Bethesda, Maryland. The University of Chicago, Chicago, Illinois. We investigated the dysregulation of DRD2 in human cancer 8Sanford Burnham-Prebys Medical Discovery Institute, La Jolla, California. and its role in tumor response to ONC201 to uncover predictive 9Massachusettes General Hospital, Boston, Massachusetts. 10Dana Farber Can- cer Institute, Boston, Massachusetts. biomarkers. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Materials and Methods Corresponding Author: Joshua E. Allen, Oncoceutics Inc., Philadelphia, PA Cell culture and reagents 19104. Phone: 1-844-662-6797; E-mail: [email protected] ONC201-resistant RKO human colon cancer cells have been doi: 10.1158/1078-0432.CCR-18-2572 generated previously using increasing concentration gradient Ó2018 American Association for Cancer Research. incubations (13). All other cell lines were obtained from the www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst December 17, 2018; DOI: 10.1158/1078-0432.CCR-18-2572 Prabhu et al. Propidium iodide staining and cell viability assays Translational Relevance Cells were treated, trypsinized, ethanol-fixed, stained with Overexpression of dopamine receptor D2 (DRD2) is asso- propidium iodide (Sigma), and analyzed by flow cytometry as ciated with a poor clinical prognosis in patients with cancer. previously described (9). Cell viability was determined as We demonstrate that DRD5, a dopamine receptor family described previously using the CellTitre-Glo reagent (Promega; member that opposes DRD2 signaling, is a negative regulator ref. 15). of tumor cell sensitivity to DRD2 antagonism. Small molecule ONC201 is the first selective antagonist of D2-like dopamine Western blot analysis receptors for clinical oncology. We report a DRD2þDRD5À Western blotting was performed as described previously (8, 11, biomarker signature that is predictive of enhanced tumor cell 18). Briefly, lysates were prepared and evaluated with protein sensitivity to ONC201 in preclinical models and is associated assay (Bio-Rad). LDS sample buffer and reducing agent (Invitro- with improved outcomes in patients treated with ONC201 gen) were added for SDS-PAGE. After transfer, primary and in a phase II clinical trial for recurrent glioblastoma secondary antibody incubations were performed, and signal was (NCT02525692). The predictive biomarker signature for detected using a Chemiluminescent Detection Kit, followed by ONC201 is under evaluation in ongoing glioma clinical trials autoradiography. and may be used to identify additional indications for ONC201, such pheochromocytoma which is now being inves- Immunohistochemistry tigated in a phase II clinical trial (NCT03034200). IHC 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 performed using the automated Leica Bond Rx system followed by dehydra- ATCC and cultured as per ATCC recommendations. Cells were tion and mounting. Following US Biomax tissue microarrays were authenticated every month by bioluminescence, growth, and used: glioblastoma (GBM; GL805B), neuroblastoma (MC602), morphologic observation. ONC201 was provided by Oncoceu- pheochromocytoma (AD2081), medulloblastoma (BC17012c), tics, Inc. and endometrial cancer (EMC961). Additionally, the BioChain FDA Standard Tissue Array (T8234701-1) was also used. Tissue GPCR profiling microarray and patient archival tumor tissue slides stained for Experimental GPCR profiling was performed utilizing the DRD2 and DRD5 were scanned (Aperio AT2 slide scanner) and PathHunter b-arrestin enzyme fragment complementation (EFC) loadedintoimageanalysissoftware(VisiopharmVIS).Wholeslide assay at DiscoverX as described previously (17, 18). PathHunter images were annotated by a pathologist to create region of interest cells were seeded in a total volume of 20 mL into white walled, (ROI) delineating tumor area and to set thresholds specific for 384-well microplates, and incubated at 37C for the appropriate DRD2- and DRD5-positive immunolabeling. Individual algo- time prior to testing. For agonist determination, cells were incu- rithms were developed to detect and stratify DRD2 or DRD5 bated with test compound to induce response. Five microliters of immunolabeling area for each TMA core or patient sample, respec- 5Â test compound was added to cells and incubated at 37Cor tively. Each algorithm identified the number of pixels stained room temperature for 90 or 180 minutes. For antagonist deter- positively for DRD2 or DRD5 within the tumor ROI. Positively- mination, cells were preincubated with test compound followed stained pixels converted to area was quantified as a percentage of by agonist challenge at the EC80 concentration (5 mLof5Â sample total tumor area. Following quantification, each sample was was added to cells and incubated at 37C or room temperature for reviewed to confirm appropriate algorithm application and quan- 30 minutes and 5 mLof6Â EC80 agonist in assay buffer was added tification. The pathologist assigned a low threshold to account for to the cells and incubated at 37C or room temperature for 90 or background signal and determine a baseline positive. 180 minutes). Final assay vehicle concentration was 1%. Assay signal was generated

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