Highly potent dopamine receptor D2 antagonist ONC206 demonstrates anti-tumorigenic activity in endometrial cancer

Abstract

Endometrial cancer (EC) is a highly obesity-driven cancer, with limited treatment options. ONC201 is an imipridone that selectively antagonizes the G protein-coupled receptors dopamine receptor D2 and D3 (DRD2/3) and activates human mitochondrial caseinolytic protease P (ClpP). It is a promising first-in-class small molecule that has been reported to have anti-neoplastic activity in various types of cancer through induction of the integrated stress response (ISR) as well as through stimulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and subsequent induction of apoptosis. ONC201 is being evaluated in Phase II clinical trials for solid tumors and hematological malignancies, including EC. ONC206 is an analog of ONC201 with nanomolar potency in Phase I clinical trials. This study evaluated the anti-tumor efficacy of ONC206 in EC cell lines and the Lkb1^fl/flp53^fl/fl genetically engineered mouse model of endometrioid EC. ONC206 revealed greater potency than ONC201 in the inhibition of proliferation in EC cell lines, with IC50 concentration ranges of 0.21-0.32 µM for ONC026 versus 2.14-3.53 µM for ONC201. ONC206 induced cellular stress, apoptosis and cell cycle G1 arrest, accompanied by inhibition of the AKT/mTOR/S6 pathways in EC cells. Diet-induced obesity accelerated tumor growth in Lkb1^fl/flp53^fl/fl mice. ONC206 inhibited EC tumor size and weight in both obese and lean mice after 4 weeks of treatment. Treatment with ONC206 led to a decrease in expression of Ki67, BCL-XL and phosphorylation of S6, as well as an increase in ClpP in endometrial tumors under both obese and lean conditions. Overall, the pre-clinical efficacy of ONC206 is promising and worthy of further exploration in clinical trials for endometrioid EC.

Keywords: ONC206; apoptosis; dopamine receptors; endometrial cancer; obesity.

Figure 1

Effect of ONC206 and ONC201 on cell proliferation in EC cells. The ECC-1 and Ishikawa cell lines were cultured in the presence of varying concentrations of ONC206 and ONC201 for 72 hours. Cell proliferation was determined by MTT assay. Based on the IC50 dose comparison, ONC206 demonstrated 14-20 times greater potency when compared to ONC201 (A). Western blotting results indicated that ONC206 induced the expression of DRD2 and DR5 in EC cells after 24 hours of treatment (B).

Figure 2

Effect of ONC206 on cell cycle progression in EC cells. The ECC-1 and Ishikawa cells were treated with ONC206 at varying doses for 24 hours. Changes in cell cycle progression were analyzed by Cellometer. ONC206 induced G0/G1 cell cycle arrest and reduced S phase and G2 phase in both EC cell lines (A). Western blotting results showed that ONC206 decreased the expression of CDK4 and CDK6 after 24 hours of treatment (B) (*P<0.05, **P<0.01).

Figure 3

ONC206 induces cellular stress in EC cell lines. The ECC-1 and Ishikawa cell lines were treated with ONC206 at the indicated doses for 4 hours. ROS was assessed by DCFDA assay. ONC206 induced cellular ROS production in a dose-dependent manner in both cell lines (A). Western blotting results showed that ONC206 increased expression of the cellular stress proteins Ero1, Perk and Bip in both EC cell lines after 24 hours of treatment (B). (*P<0.05, **P<0.01).

Figure 4

ONC206 significantly decreased tumor weight in the Lkb1^fl/flp53^fl/fl EC mouse model. Lkb1^fl/flp53^fl/fl mice were fed a HFD (obese) or LFD (lean) starting at 3 weeks of age. Diet-induced obesity significantly increased the body weights of Lkb1^fl/flp53^fl/fl mice (A). The mice were treated with ONC206 (125 mg/kg, oral gavage, weekly) or vehicle for 4 weeks, beginning 8 weeks after tumor induction. ONC206 significantly inhibited tumor weight under obese and lean conditions (B). IHC results showed that ONC206 treatment reduced the expression of Ki67 and increased the expression of DRD2 and ClpP in endometrial tumor tissues (C). (*P<0.05, **P<0.01).

Figure 5

ONC206 induced apoptosis in vitro and in vivo. Cells were treated with ONC206 at the indicated doses for 24 hours and then analyzed for expression of Annexin V by Cellometer. ONC206 increased the expression of Annexin V in a dose-dependent manner in both cell lines (A). Western blotting results showed that ONC206 significantly reduced the expression of MCL-1 and BCL-XL in both cell lines (B). IHC results demonstrated that ONC206 decreased the expression of BCL-XL in endometrial tumor tissues (C). (*P<0.05, **P<0.01).

Figure 6

ONC206 inhibited mTOR/S6 pathway in vitro and in vivo. The ECC-1 and Ishikawa cell lines were treated with ONC206 at varying doses for 24 hours. Western blotting results demonstrate that ONC206 decreased phosphorylation of S6 and increased phosphorylation of AKT in both cell lines (A). Treatment with ONC206 for 4 weeks significantly decreased the expression of phosphorylated S6 in endometrial tumor tissues of Lkb1^fl/flp53^fl/fl mice (B).