Epigenetic Priming By Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-Cell Lymphoma
Authors: Ward George , Zhang Zhiqiang , Jueliger Simone , Potapov Ilya , Davis Matthew P , Boxall Adam , Taylor Jason A , Keer Harold N. , Biondo Andrea , Lyons John F , Sims Martin , Smyth Tomoko 
Published: December 2023

Background:Durable single-agent responses with the IAP antagonist, tolinapant (ASTX660), have been reported in peripheral T-cell lymphoma (PTCL) and cutaneous TCL (CTCL) patients (NCT02503423, Michot et al., EHA 2022). We have found that tolinapant has the potential to drive immunogenic form of cell death (ICD), such as necroptosis, in preclinical models of TCL (Ferrari et al., Blood Adv. 2021). Here, we further explored this by modulating key necrosome components in TCL cell lines and analysing changes in cell death signalling. We hypothesised the epigenetic mechanisms that limit induction of ICD could be reversed by combining tolinapant and hypomethylating agents, such as decitabine. Our biomarker-led approach demonstrated that this novel combination could overcome inherent resistance to a form of cell death that can engage a strong immune response.
Materials and Methods: Key necrosome components were modified in human and mouse TCL cell lines by CRISPR knockout and activation, and effects of tolinapant on their viability measured in vitro. Promoter methylation was assessed by pyrosequencing following bisulfite treatment (EpigenDx). Effects of tolinapant and decitabine, as single-agents and in combination, on cytokine/chemokine release were assessed using Meso Scale Discovery (MSD) or Luminex assay. ICD biomarkers in cells and tumor lysates were analysed by ELISAs, MSD assays, qPCR and Western blotting. In vivo activity of tolinapant and decitabine were tested in caspase-8 knockout EL4 cells in a syngeneic setting. PTCL patient plasma samples were analysed by Human ExplorerMAP™ v. 1.0 (Luminex xMAP® technology).
Results: Loss of necrosome components or necrosome inactivation led to reduced sensitivity of TCL cells to tolinapant. We confirmed decitabine treatment resulted in re-expression of necrosome proteins (e.g., RIPK3) and cancer testis antigens. Combination of decitabine and tolinapant led to increased lytic cell death over monotherapies, and this was accompanied by increased levels of interferon signalling and ICD biomarkers (e.g., HMGB1). Tolinapant treatment alone, or in combination with decitabine, led to elevated chemokine release from dying TCL cells. These observations indicate activation of necroptosis. In vivo, the combination treatment prolonged survival of mice bearing caspase-8 knockout EL4 tumors while monotherapies had limited effects. We also report that IP-10 (CXCL10) levels were increased in plasma samples of tolinapant-treated PTCL patients, as well as other plasma proteins, implying immune activation.
Conclusions: We demonstrate that tolinapant acts as an immune modulator via promoting ICD, and this can be enhanced by epigenetic reprogramming with decitabine. These findings provide a rationale to support the on-going combination study of tolinapant with ASTX727 (oral decitabine and cedazuridine) that recently started in PTCL (ASCERTAIN-P, NCT05403450). It has also identified putative biomarkers of immunomodulatory activity that could be utilized in clinic.