Identification of TRAIL-inducing compounds highlights small molecule ONC201/TIC10 as a unique anti-cancer agent that activates the TRAIL pathway

Abstract

Background: We previously reported the identification of ONC201/TIC10, a novel small molecule inducer of the human TRAIL gene that improves efficacy-limiting properties of recombinant TRAIL and is in clinical trials in advanced cancers based on its promising safety and antitumor efficacy in several preclinical models.

Methods: We performed a high throughput luciferase reporter screen using the NCI Diversity Set II to identify TRAIL-inducing compounds.

Results: Small molecule-mediated induction of TRAIL reporter activity was relatively modest and the majority of the hit compounds induced low levels of TRAIL upregulation. Among the candidate TRAIL-inducing compounds, TIC9 and ONC201/TIC10 induced sustained TRAIL upregulation and apoptosis in tumor cells in vitro and in vivo. However, ONC201/TIC10 potentiated tumor cell death while sparing normal cells, unlike TIC9, and lacked genotoxicity in normal fibroblasts. Investigating the effects of TRAIL-inducing compounds on cell signaling pathways revealed that TIC9 and ONC201/TIC10, which are the most potent inducers of cell death, exclusively activate Foxo3a through inactivation of Akt/ERK to upregulate TRAIL and its pro-apoptotic death receptor DR5.

Conclusion: These studies reveal the selective activity of ONC201/TIC10 that led to its selection as a lead compound for this novel class of antitumor agents and suggest that ONC201/TIC10 is a unique inducer of the TRAIL pathway through its concomitant regulation of the TRAIL ligand and its death receptor DR5.

Figure 1

Identification of TRAIL-inducing compounds by high-throughput luciferase reporter screen. (a) Plot of reporter induction versus signal for screen conducted with the NCI Diversity Set II at 1 μM in HCT116 Bax^−/− cells that stably express a luciferase gene reporter construct under transcriptional control of the first 504 base pairs of the human TRAIL gene promoter. Induction is expressed as luminescence relative to DMSO-treated cells. The horizontal red line represents the cutoff for selection. (b) Validation of reporter induction for candidate screen hits in HCT116 Bax^−/− cells at multiple doses and time points. Reporter signal is expressed relative to DMSO-treated cells and normalized to cell viability. The horizontal red line represents the cutoff for selection. Data represent an average of three replicates. The horizontal red line represents the cutoff for selection. (C) Molecular structures of the selected TRAIL-inducing compounds.

Figure 2

TIC9 and ONC201/TIC10 induce TRAIL gene transcription and surface TRAIL upregulation in tumor cells in vitro. (a) Reporter activity in HCT116 Bax^−/− cells that stably express a luciferase gene reporter construct under transcriptional control of the first 504 base pairs of the human TRAIL gene promoter (n = 3). Reporter signal is expressed relative to DMSO-treated cells and normalized to cell viability. (b) RT-qPCR analysis of TRAIL gene transcription in response to TRAIL-inducing compounds (TICs) (5 μM) in HCT116 p53^−/− cells (n = 4, 48 hr). (c) Flow cytometry analysis of surface TRAIL in response to TICs (5 μM) in HCT116 p53^−/− cells (n = 3, 72 hr). *P < .05 by student’s two-tailed t test. Error bars represent standard deviation.

Figure 3

TIC4, TIC9, and ONC201/TIC10 upregulate TRAIL in vivo. (a) RT-qPCR analysis of HCT116 p53^−/− tumor xenograft in athymic nude mice harvested 2 days following a single intravenous dose of DMSO, TIC4, TIC9, or ONC201/TIC10 (25 mg/kg). *P < .05 by student’s two-tailed t test. (b) IHC analysis of TRAIL protein levels in HCT116 p53^−/− tumor xenograft in athymic nude mice harvested following a single intravenous dose of DMSO, TIC4, TIC9, or ONC201/TIC10 (25 mg/kg). (c) Exemplary images and (d) quantification of TUNEL staining in TIC-treated xenograft tumors described in (a). (e) Bioluminescent imaging of luciferase-infected HCT116 subcutaneous xenografts following a single intraperitoneal dose of TIC9 and ONC201/TIC10 (100 mg/kg) on day 0. Quantification of tumor signals shown in right panel (n = 6). Error bars represent standard deviation.

Figure 4

TIC9 and ONC201/TIC10 induce tumor cell death and ONC201/TIC10-induced cell death is tumor-selective. (a) Sub-G1 DNA content of HCT116 p53^−/− cells treated with TICs for indicated time periods (1 μM, n = 3). (b) Cell cycle profiles of HCT116 p53^−/− cells treated with TICs (5 μM, 72 hr). (c) Sub-G1 DNA content of HCT116 p53^−/− cells treated with TICs (72 hrs, 5 μM, n = 3). (d) Sub-G1 DNA content of HFF normal human fibroblasts treated with TICs (72 hr, 5 μM, n = 3). (e) Cell viability assay of HCT116 or HFF cells treated with ONC201/TIC10 at the indicated doses (72 hr, n = 3). Error bars represent standard deviation.

Figure 5

ONC201/TIC10 is not genotoxic to normal human fibroblasts. (a) Bright field microscopy images of HFF fibroblasts following 72 hr incubation with DMSO, ONC201/TIC10, or doxorubicin (1 μM) as indicated. (b) Immunofluorescence imaging of gamma H2AX (green) and nuclear counterstain (blue) in HFF fibroblasts treated as described in (a).

Figure 6

Selection of ONC201/TIC10 as a lead TRAIL-inducing compound. Prioritization assays are shown for the process of selecting ONC201/TIC10 as a lead compound.

Figure 7

ONC201/TIC10 and TIC9 exclusively affect Akt/ERK/Foxo3a. Western blot analysis of HCT116 p53^−/− cells treated with TICs in HCT116 p53^−/− cells (5 μM, 60 hr).