ONC201 Demonstrates Antitumor Effects in Both Triple-Negative and Non-Triple-Negative Breast Cancers through TRAIL-Dependent and TRAIL-Independent Mechanisms

Abstract

Breast cancer is a major cause of cancer-related death. TNF-related apoptosis-inducing ligand (TRAIL) has been of interest as a cancer therapeutic, but only a subset of triple-negative breast cancers (TNBC) is sensitive to TRAIL. The small-molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here, we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n = 13). A subset of TNBC and non-TNBC cells succumbs to ONC201-induced cell death. In 2 of 8 TNBC cell lines, ONC201 treatment induces caspase-8 cleavage and cell death that is blocked by TRAIL-neutralizing antibody RIK2. The proapoptotic effect of ONC201 translates to in vivo efficacy in the MDA-MB-468 xenograft model. In most TNBC lines tested (6/8), ONC201 has an antiproliferative effect but does not induce apoptosis. ONC201 decreases cyclin D1 expression and causes an accumulation of cells in the G₁ phase of the cell cycle. pRb expression is associated with sensitivity to the antiproliferative effects of ONC201, and the compound synergizes with taxanes in less sensitive cells. All non-TNBC cells (n = 5) are growth inhibited following ONC201 treatment, and unlike what has been observed with TRAIL, a subset (n = 2) shows PARP cleavage. In these cells, cell death induced by ONC201 is TRAIL independent. Our data demonstrate that ONC201 has potent antiproliferative and proapoptotic effects in a broad range of breast cancer subtypes, through TRAIL-dependent and TRAIL-independent mechanisms. These findings develop a preclinical rationale for developing ONC201 as a single agent and/or in combination with approved therapies in breast cancer. Mol Cancer Ther; 16(7); 1290-8. ©2017 AACR.

Figure 1. ONC201 induces cell death in TNBC and non-TNBC cells

A) Annexin-V/PI double positive cells were quantified using flow cytometry following a 72 hour treatment with a vehicle control or 10 μM ONC201 (n=2 experiments for each cell line). B) Western blot of TNBC cells treated with a vehicle control or 10 μM ONC201 for 72 hours to compare PARP cleavage. ns: p≥0.05; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 2. The pro-apoptotic effects of ONC201 in some TNBC cells involve the extrinsic pathway, are TRAIL-dependent, and translate to efficacy in the MDA-MB-468 breast cancer xenograft model

A) Western blot of MDA-MB-468 and SUM149PT TNBC cells treated with a vehicle control or 10 μM ONC201 for 72 hours to show caspase-8 cleavage. B) Annexin-V/PI double positive cells were quantified using flow cytometry following a 72 hour treatment with a vehicle control or ONC201 (MDA-MB-468: 5 μM, SUM149PT: 10 μM), with or without 1 μg/mL RIK2 TRAIL blocking antibody (n=2 experiments for each cell line). C) Fold change tumor volume measured over time in nude mice bearing MDA-MB-468 xenografted tumors treated orally with a vehicle control (n=3), 50 mg/kg ONC201 once per week (n=3), or 50 mg/kg ONC201 three times per week (n=4). D) Comparison of fold change tumor volume in mice treated with a vehicle control, 50 mg/kg ONC201 once per week, or 50mg/kg ONC201 three times per week on day 45 of the experiment. ns: p≥0.05; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 3. Differential sensitives to anti-proliferative effects of ONC201 are observed among TNBC cells that do not undergo apoptosis

A) Dose response curves of TNBC cells treated with ONC201 for 72 hours. B) Trypan blue exclusion used to determine viable cell count in TNBC cells over time following treatment with a vehicle control or 10 μM ONC201. C) Percent BrdU-positive cells over time in TNBC cells following treatment with a vehicle control or 10 μM ONC201. ns: p≥0.05; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 4. ONC201 activates an integrated stress response, decreases cyclin D1 expression, and causes an accumulation of cells in G₁ in TNBC cells sensitive to the compounds anti-proliferative effects

A) Western blot analysis of MDA-MB-231 cells for EIF2α phosphorylation, ATF4 expression, and cyclin D1 expression and following treatment with a vehicle control, 5 μM, or 10 μM ONC201 for 72 hours. Densitometry was performed using NIH ImageJ software. The ratio represents the intensity of the phospho-EIF2α band divided by the intensity of the total EIF2α band. B) Quantification of cells in G₀/G₁, S, and G₂ phases of the cell cycle using flow cytometric analysis of propidium iodide stained MDA-MB-231 cells at 24 and 48 hours following treatment with a vehicle control or 10 μM ONC201. ns: p≥0.05; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 5. Rb levels during ONC201 induced growth arrest in the G₁ phase of the cell cycle

A) Quantification of cells in G₀/G₁, S, and G₂ phases of the cell cycle using flow cytometric analysis of propidium iodide stained MDA-MB-436 cells at 24 and 48 hours following treatment with a vehicle control or 10 μM ONC201. B) Western blot analysis of baseline pRb expression in TNBC cells. C) Western blot analysis of MDA-MB-231 cells treated with a vehicle control or 10 μM ONC201 over time probed to examine changes in levels of phosphorylated and total Rb. D) Quantification of cells in G₀/G₁, S, and G₂ phases of the cell cycle using flow cytometric analysis of propidium iodide stained MDA-MB-436 cells at 72 hours following treatment with a vehicle control or 10 μM ONC201.ns: p≥0.05; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 6. The pro-apoptotic effects of ONC201 in non-TNBC cells involve caspase 8 cleavage, but are TRAIL-independent

A) Immunoblot analysis of SKBR3 and MCF7 cells treated with a vehicle control or 10μM ONC201 for 72 hours. B) Geometric mean fluorescence intensity of TRAIL and DR5 surface staining was determined in MDA-MB-231 and MCF7 cells treated with a vehicle control or 5 or 10μM ONC201 for 72 hours. C) Annexin-V/PI double positive cells were quantified in SKBR3 using flow cytometry following a 72 hour treatment with a vehicle control or 10μM ONC201 in the presence or absence of 1 ug/mL RIK2 TRAIL blocking antibody. D) Immunoblot of MCF7 cells treated with a vehicle control or 10μM ONC201 in the presence or absence of 1 ug/mL RIK2 TRAIL blocking antibody.