ONC201 Induces Apoptosis in Cutaneous T-Cell Lymphoma Cells through a Mechanism That Involves the Integrated Stress Response and Inactivation of Jak/Stat Signaling
Authors: Duvic Madeleine , Zhang Xiang , Ni Xiao , Langridge Timothy , Tarapore Rohinton , Allen Josh 
Published: October 2019

Background: ONC201 (also called TIC10) is an orally active, first-in-class small molecule that is in phase II clinical trials for advanced cancers based on its ability to activate apoptosis in tumor cells, but not normal cells, in a p53-independent manner. Recent studies have implicated the integrated stress response as an early stage mechanism of ONC201 that may lead to the previously observed downstream anti-cancer effects. This study was conducted to evaluate the anti-tumor activity of ONC201 on Cutaneous T-Cell Lymphoma (CTCL) cell lines and Sézary cells.

Methods: We treated 8 CTCL cell lines (H9, HH, Hut78, Mac2A, MJ, MyLA, Pb2b and SeAx), peripheral blood mononuclear cells (PBMC) from 5 Sézary syndrome (SS) patients and 6 healthy donors with ONC201. MTS viability Assay was used to assess the anti-proliferation effect. Apoptosis was assessed using Annexin V FITC/PI staining and sub-G1 analysis by flow cytometry. Protein expression by western blotting was analyzed in 3 CTCL cell lines (HH, Hut78, MJ) and in PBMCs from 2 SS patients treated for 72 hours with ONC201including Activating Transcription Factor 4 (ATF4), interferon regulatory factor 7 (IRF7/pIRF7), Signal transducer and activator of transcription 3 (STAT3), Janus Kinase 3 (JAK3/pJAK3) and NF-κB family members (p65, RelB, c-Rel and p105).

Results: MTS viability assay showed pronounced cell growth inhibition, significantly increasing in a time-dependent manner in H9, HH, Hut78, Mac2A, MJ, MyLa, Pb2b, and SeAx lines after a 96-hour incubation within a very narrow efficacy threshold ranging from 1.25 to 10µM (n = 8, p < 0.05). To determine whether growth inhibition of ONC201 is due to cell-cycle arrest and/or to apoptosis in CTCL cell lines, sub-G1 analysis by flow cytometry was measured in 3 CTCL cell lines (HH, Hut78 and MJ). The percentages of HH, Hut78 and MJ cells with sub-G1 population increased, suggesting that cells are undergoing apoptosis (n = 3, p < 0.05). Induction of apoptosis was further confirmed by Annexin V FITC/PI staining, revealing dose and time dependent apoptosis induction in all 8 cell lines (Figure 1). To confirm cell line results in refractory ex vivo samples, we tested the pro-apoptotic effects of ONC201 on PBMCs from 5 SS patients who had high circulating CD4+CD26- malignant T-cells compared with PBMCs from 6 healthy donors. ONC201 induced significant levels of apoptosis in PBMCs from SS patients, but not in normal PBMCs (Figure 1, p < 0.001). Western blot analysis revealed that ONC201 increased the expression of ATF4, a hallmark of the integrated stress response and a negative regulator of IRF7. ONC201 decreased expression of IRF7/pIRF7 and down-regulated JAK3/pJAK3 and STAT3 expression in CTCL cell lines and PBMCs from SS patients. ONC201 also decreased the protein expression of NF-κB family members ( p65, RelB, c-Rel and p105) that can cause resistance to apoptosis in CTCL cells.

Conclusions: ONC201 appears to be active as a single oral agent. It may impact key signaling pathways in CTCL preclinical models by inhibiting proliferation and inducing apoptosis through a mechanism that involves the integrated stress response, leading to inactivation of JAK/STAT signaling and down-regulation of the NFκB pathway.

Figure 1. ONC201 induces apoptosis in CTCL cell lines and PBMCs from SS patients Figure 1. ONC201 induces apoptosis in CTCL cell lines and PBMCs from SS patients

Disclosures

Duvic: Eisai: Research Funding; MiRagen Therapeutics: Consultancy; Cell Medica Ltd: Consultancy; Tetralogics SHAPE: Research Funding; Soligenics: Research Funding; Array Biopharma: Consultancy; Innate Pharma: Research Funding; Allos (spectrum): Research Funding; Rhizen Pharma: Research Funding; Spatz Foundation: Research Funding; Oncoceutics: Research Funding; Huya Bioscience Int'l: Consultancy; Therakos: Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Most drugs for ctcl are non approved. This drug is a small molecule and is not approved for any cancer. Tarapore:Oncoceutics, Inc: Employment, Equity Ownership. Allen:Oncoceutics, Inc: Employment, Equity Ownership.