Cell experiment:

Floating and adherent cells are analyzed on a Coulter-Beckman Elite Epics cytometer. For surface TRAIL experiments, adherent cells are harvested by brief trypsinization, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, incubated with an anti-TRAIL antibody at 1:250 overnight, washed and incubated with anti-rabbit Alexa Fluor 488 for 30 min, and analyzed. Cells are gated on forward and side scatter to eliminate debris and dead cells from the analysis. Surface TRAIL data are expressed as median fluorescence intensity relative to that of control samples unless indicated otherwise. Surface DR5 is analyzed similarly with an antibody from Imgenex. For sub-G1content and cell cycle profile analysis, all cells are pelleted and ethanol-fixed, followed by staining with propidium iodide in the presence of RNase. Cell viability assays are carried out in 96-well black-walled clear-bottom plates with CellTiter-Glo[1].

Animal experiment:

Mice[1] For subcutaneous xenografts, 4- to 6-week-old female athymic nu/nu mice are inoculated with 1×106 cells (2.5×106 for T98G) of the indicated cell lines in each rear flank as a 200-μL suspension of 1:1 Matrigel (BD)/PBS. All subcutaneous tumors are allowed to establish for 1 to 4 weeks after injection until reaching a volume of ~125 mm3 before treatment initiation. All intraperitoneal and intravenous injections are given at a total volume of 200 μL. Oral formulations of TIC10 are administered by oral gavage and given as a 200 μL suspension containing 20% Cremophor EL, 10% DMSO, and 70% PBS. Tumors are monitored with digital calipers at indicated time points. All subcutaneous tumors are allowed to establish for 1-4 weeks post-injection until reaching a volume of ~125 mm3 before treatment initiation. Relief of tumor burden is monitored for 3 weeks after disappearance of the tumor and confirmed by visual inspection after euthanasia.

ONC201 is a first-in-class small chemical that activates p53-independent apoptosis with IC50 value less than 2.5 μM in lymphoma and leukemia cells. [1]

ONC201 induced apoptosis in stem and progenitor AML cells andabolishthe engraftment of leukemic stem cells in mice while sparing normal bone marrow cells. ONC201 induced changes in gene expression similar to those induced by the unfolded protein response (UPR) and integrated stress responses (ISRs). ONC201-treated hematopoietic cells promoted apoptosis and did not depend on increased phosphorylation of eIF2a. ONC201 also forbid mammalian target of rapamycin complex 1 (mTORC1) signaling, likely through ATF4-mediated induction of the mTORC1 inhibitor DDIT4. Overexpression of BCL-2 protected against ONC201-induced apoptosis, and the combination of ONC201 and the BCL-2 antagonist ABT-199 synergistically increased apoptosis. [1]

In solid tumors, ONC201 caused late-stage induction of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) and promoted the transcription of TRAIL gene. ONC201 has substantial antitumor activity in preclinical models in various advanced solid tumors with infrequent oral dosing and without toxicity in normal cells in culture and in vivo [2]. Preclinical studies demonstrated broad synergism of ONC201 with established anticancer therapies, including the depletion of colorectal cancer stem cells [3].

ONC201 induced p53-independent apoptosis and cell cycle arrest in acute myeloid leukemia (AML) and mantle cell lymphoma (MCL) samples from patients; these embraced samples from patients with genetic abnormalities associated with poor prognosis or cells that had produced resistance to the nongenotoxic drugs ibrutinib and bortezomib. [1]

References:

[1].ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies.Sci Signal. 2016 Feb 16;9(415):ra17.

[2].Dual in activation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects. Sci. Transl. Med. 5, 171ra117 (2013).

[3].Genetic and pharmacological screens converge in identifying FLIP, BCL2 and IAP proteins as key regulators of sensitivity to the TRAIL-inducing anti-cancer agent ONC201/TIC10. Cancer Res. 75, 1668–1674 (2015).