Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism

Abstract

Purpose: Dopamine receptor D2 (DRD2) is a G protein-coupled receptor antagonized by ONC201, an anticancer small molecule in clinical trials for high-grade gliomas and other malignancies. DRD5 is a dopamine receptor family member that opposes DRD2 signaling. We investigated the expression of these dopamine receptors in cancer and their influence on tumor cell sensitivity to ONC201.

Experimental design: The Cancer Genome Atlas was used to determine DRD2/DRD5 expression broadly across human cancers. Cell viability assays were performed with ONC201 in >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell lines. IHC staining of DRD2/DRD5 was performed on tissue microarrays and archival tumor tissues of glioblastoma patients treated with ONC201. Whole exome sequencing was performed in RKO cells with and without acquired ONC201 resistance. Wild-type and mutant DRD5 constructs were generated for overexpression studies.

Results: DRD2 overexpression broadly occurs across tumor types and is associated with a poor prognosis. Whole exome sequencing of cancer cells with acquired resistance to ONC201 revealed a de novo Q366R mutation in the DRD5 gene. Expression of Q366R DRD5 was sufficient to induce tumor cell apoptosis, consistent with a gain-of-function. DRD5 overexpression in glioblastoma cells enhanced DRD2/DRD5 heterodimers and DRD5 expression was inversely correlated with innate tumor cell sensitivity to ONC201. Investigation of archival tumor samples from patients with recurrent glioblastoma treated with ONC201 revealed that low DRD5 expression was associated with relatively superior clinical outcomes.

Conclusions: These results implicate DRD5 as a negative regulator of DRD2 signaling and tumor sensitivity to ONC201 DRD2 antagonism.

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 DRD2 log(expression + 1) versus normalized survival values for different cancers in TCGA (P =.0348, Fisher’s exact test). (C) Quantification and (D) exemplary images from DRD2 IHC analysis of tissue microarrays in GBM (n=35), neuroblastoma (n=25), medulloblastoma (n=20), endometrial cancer (n=32) and pheochromocytoma (n=30) with corresponding normal tissue. Dotted lines represents average expression for each tumor type. Quantification for normal tissue could not be performed due to low number of samples. Data is presented as DRD2-positive staining as a percentage of total tumor area.

Figure 2.. DRD2 dysregulation in human glioma and concordance with tumor response to DRD2 antagonism by ONC201.

(A) Distribution of DEMETER essentiality scores for DRD2 by primary tumor type. (B) Expression of dopamine receptors in GBM specimens by RNA-seq from TCGA by primary versus secondary GBM. (C) DRD2 expression in glioblastoma versus overall survival in patients. (D) ONC201 GI50 in NCI60 GBM cell lines versus DRD2 expression (48 h). (E) Scatter plot showing the correlation between brain cancer cell line-matched ONC201 AUC values and DRD2 DEMETER essentiality scores, with correlation and p-value calculated using Spearman correlation test.

Figure 3.. DRD5 is an inversely correlated predictive biomarker of tumor cell sensitivity to ONC201.

(A) DRD5 missense mutation identified by whole exome sequencing in RKO cells with acquired resistance to ONC201. Points are colored based on the type of genomic event. Overlapping mutation in DRD5 among 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 wild-type or Q366R DRD5 constructs. Quantitation of DRD2 monomer and dimer expression normalized to GAPDH is shown on the right. (E) Western blot analysis of PARP cleavage in HCT116 cancer cells treated with ONC201 with overexpression of wild-type or Q366R DRD5. Quantitation of cleaved PARP normalized to total PARP and Ran is shown at the bottom. (F) Expression of DRD5 in human tumor samples in TCGA. (G) ONC201 GI50 of NCI60 cells categorized by DRD5 mRNA expression using z-score (48 h). (H) ONC201 efficacy distributions for DRD2+/DRD5- and DRD2-/DRD5- samples in the GDSC panel (P = .0037, KS Test, 72 h). Cutoffs for DRD2 expression were z-scores of 1 and −1. Cutoffs for DRD5 were z-scores of 0.5 and −0.5.

Figure 4.. Intratumoral DRD5 expression is associated with clinical outcomes of ONC201-treated adult recurrent glioblastoma patients.

(A) DRD5 expression by IHC analysis in archival tumor samples categorized by PFS>5 months (n=3) or PFS<5 months (n=12) of ONC201-treated recurrent GBM patients (625mg q3w PO). Data is presented as DRD5-positive staining as a percentage of total tumor area. (B) Exemplary DRD5 expression by IHC analysis in archival tumor specimens from ONC201-treated recurrent GBM patients who experience an objective response or progressive disease. (C) Overall survival of ONC201-treated recurrent GBM patients who had archival tumor specimens that were DRD5- versus DRD5+ by IHC analysis. Gray circles indicate a censoring event for followup. (D) Thalamic and (E) parietal lobe GBM lesions before and 17 months after 625mg q3w ONC201. (F) Overall tumor burden over time in responding recurrent GBM patient treated with ONC201.