Akt; ERK

ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death [1].

In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses [1].

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TIC10 and TRAIL treatment causes tumor regression in the HCT116 p53−/− xenograft to a comparable extent when both are administered as multiple doses. TIC10 also induces regression of MDA-MB-231 human triple-negative breast cancer xenografts, whereas TRAIL-treated tumors progressed. In DLD-1 colon cancer xenografts, TIC10 induces tumor stasis one week after treatment, whereas TRAIL-treated tumors advance after a single dose. The SW480 xenograft also exhibits a sustained regression after receiving a single dose of TIC10, and this effect is seen whether the drug is administered orally or intraperitoneally. This suggests that TIC10 has a favorable oral bioavailability. TIC10 causes tumor-specific cell death by TRAIL-mediated direct and bystander effects. TIC10 is an effective antitumor agent against orthotopic human glioblastoma multiforme tumors. [2]

siRNA kinase library screen[1]
HCT116 p53−/− cells were transfected at 20,000 cells/well in 96-well plates. Cells were transfected with the Stealth RNAi human kinase library using RNAiMax in Optimem. Scramble and AllStars Hs Cell Death Control siRNA were used as negative and positive controls, respectively. Transfection was carried out overnight and the following day complete media containing ONC201 or DMSO was added to the plates following removal of the transfection media. CellTiterGlo analysis was performed according to the manufacturer’s instructions at indicated time points.

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ChIP assays [2]
ChIP assays were carried out as previously described for the TRAIL promoter with a ChIP-grade antibody for Foxo3a or an equivalent concentration of rabbit immunoglobulin G as a nonspecific control.

Small molecule synergy screen[1]
Procedures were performed with a Biomek 2000 robot using pin tools for drug treatment. Cells were seeded at 5×104 cells/mL in 96-well black-wall plates and treatment was performed 12 hours later. Combinatorial activity was initially assessed by calculating the difference between the observed activity with the combination and the sum of the monoagent activities.

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Cell-based assays[1]
Cell TiterGlo, Western blot analysis, and cell cycle flow cytometry analysis were performed as previously described. Cell surface TRAIL and DR5 were assessed following fixation with 4% paraformaldehyde in PBS for 20 minutes, rinsed in PBS, incubated overnight at 1:200 with anti-TRAIL or anti-DR5 antibodies, rinsed in PBS, incubated with fluorophore-conjugated secondary antibodies at 1:250 for 30 minutes, and analyzed by flow cytometry. pERK was assessed with an antibody for p-T202/Y204 (Cell Signaling) and pAkt was assessed with antibody for pT308.

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Cells were treated with 10 μM ONC201 or DMSO for 24 h.
Colony formation assays [2]
The indicated cell lines were plated at 500 cells per well and allowed to adhere, and then treated the next day in fresh complete medium. At 3 days after treatment, the medium was replaced with drug-free medium and cells were propagated for 10 days, with fresh medium given once every 3 days. At the end of the 10-day period, cells were washed in PBS, fixed with methanol, stained with Coomassie blue, rinsed, and dried for quantification.

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Western blot analysis [2]
Western blot analysis was conducted as previously described with NuPAGE 4 to 12% bis-tris gel and visualized with SuperSignal West Femto and x-ray film. Densitometry was performed with NIH ImageJ. Nuclear and cytoplasmic extracts were prepared with a cytoplasmic lysis buffer (10 mM Hepes, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol) followed by a nuclear lysis buffer (20 mM Hepes, 420 mM NaCl, 1.5 mM MgCl2, 250 μM EDTA, 25% glycerol). For all lysis buffers, fresh protease inhibitor and 1 mM sodium orthovanadate were added immediately before use.

6-week-old athymic nude mice were obtained from Charles River Laboratories. 107 HepG2 cells in 200 µL (1:1, PBS: Matrigel) were injected into each rear flank. Measurable tumors were assessed 1 week later. Treatment was then initiated as indicated. Sorafenib and ONC201 were administered as 100 µL solutions by oral gavage. ONC201 was administered 12 hours following sorafenib treatment. Tumor volume was assessed with digital calipers and calculated as a spheroid. Tumor dimensions and body weights were assessed twice a week. IHC (Vector Labs) and TUNEL (Millipore) analyses were performed as previously described [1].

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For subcutaneous xenografts, 4- to 6-week-old female athymic nu/nu mice (Charles River Laboratories) were 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 were allowed to establish for 1 to 4 weeks after injection until reaching a volume of ~125 mm3 before treatment initiation.[2]

[1]. Cancer Res.2015 Apr 15;75(8):1668-74;
[2]. Sci Transl Med.2013 Feb 6;5(171):171ra17.

NC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses. [1]

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Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal-regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL. [2]

C24H28CL2N4O

459.411323547363

458.164

C, 62.75; H, 6.14; Cl, 15.43; N, 12.20; O, 3.48

1638178-82-1

41276-02-2 (isomer);1616632-77-9;1638178-82-1 (HCl);1777785-71-3 (HBr); 2007141-57-1 (2HBr);

121596510

Solid powder

2

3

4

31

693

0

Cl.Cl.O=C1C2CN(CC3C=CC=CC=3)CCC=2N2CCN=C2N1CC1C=CC=CC=1C

HKBXPCQCBQFDML-UHFFFAOYSA-N

InChI=1S/C24H26N4O.2ClH/c1-18-7-5-6-10-20(18)16-28-23(29)21-17-26(15-19-8-3-2-4-9-19)13-11-22(21)27-14-12-25-24(27)28/h2-10H,11-17H2,1H32*1H

7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-one dihydrochloride

ONC-201 Dihydrochloride, ONC-201 HCl, ONC201; ONC 201 ONC-201; NSC350625; NSC-350625; NSC 350625; ONC-201 Dihydrochloride; 1638178-82-1; ONC201 HCl; 53VG71J90J; 7-Benzyl-4-(2-methylbenzyl)-2,4,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one dihydrochloride; 11-benzyl-7-[(2-methylphenyl)methyl]-2,5,7,11-tetrazatricyclo[7.4.0.02,6]trideca-1(9),5-dien-8-one;dihydrochloride; Imidazo(1,2-a)pyrido(3,4-E)pyrimidin-5(1H)-one, 2,4,6,7,8,9-hexahydro-4-((2-methylphenyl)methyl)-7-(phenylmethyl)-, hydrochloride (1:2); Imidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one, 2,4,6,7,8,9-hexahydro-4-[(2-methylphenyl)methyl]-7-(phenylmethyl)-, hydrochloride (1:2); imipridone; TIC10; TIC 10; TIC-10 TRAIL inducing compound 10.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO; > 10 mM

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)