ONC201 Induces Apoptosis in Cutaneous T-Cell Lymphoma Cells through a Mechanism That Involves the Integrated Stress Response and Inactivation of Jak/Stat Signaling

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 Sezary cells. Methods: We treated 8 CTCL cell lines (H9, HH, Hut78, Mac2A, MJ, MyLA, Pb2b and SeAx), peripheral blood mononuclear cells (PBMC) from 5 Sezary 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 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 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. 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 Int9l: Consultancy; Therakos: Research Funding, Speakers Bureau; Celgene: Membership on an entity9s Board of Directors or advisory committees; Seattle Genetics: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity9s 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.