pCMV delta R8.2

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that leads to early activation of the integrated stress response. Binding and reporter gene assays indicate that ONC201 is a selective antagonist of dopamine D2-like receptors, specifically DRD2 and DRD3. Here, researchers demonstrate that DRD2 is expressed in different cancer cell types in a cell type-specific manner. DRD3, on the other hand, is often undetectable. Overexpression of DRD2 in cells with low DRD2 levels increases ONC201-induced PARP cleavage that precedes and correlates with the ONC201-induced increase in CHOP mRNA expression. On the other hand, knockdown of DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abolish the anticancer effects of ONC201. Transient DRD2 knockdown in HCT116 cells activated an integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduces cell viability. This study therefore demonstrates that disrupting dopamine receptor expression and activity may produce cytotoxic effects that may be attributable, at least in part, to activation of the integrated stress response. On the other hand, the anticancer activity of ONC201 exceeds its ability to antagonize DRD2, which may be due to ONC201's ability to activate other pathways independent of DRD2.

In this study, five different lentiviral plasmid shRNA constructs of DRD2 were amplified and the DNA was isolated using a plasmid kit. Lentiviral particles were generated by transfecting HEK293T cells with shDRD2 DNA, packaging plasmid pCMV-VSV-G, and pCMV delta R8.2 at a 2:1:1 ratio. After 72 hours, lentiviral particles were collected, diluted with an equal volume of culture medium, and added to HCT116 cell cultures. Stable transfectants were selected with 1 μg/ml puromycin. Knockdown efficiency was assessed by analyzing DRD2 mRNA expression by qRT-PCR.

Figure 1. qRT-PCR analysis of DRD1 and DRD2 mRNA expression was performed to verify knockdown and monitor potential compensatory overexpression of DRD1 receptors. (Kline, Christina Leah B., et al., 2018)