The combination of the IAP antagonist, tolinapant and hypomethylating agents (HMA) is highly synergistic in in vitro models of T-cell lymphoma (TCL).

Abstract

e15088 Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent antagonist of inhibitors of apoptosis proteins (cIAP1/2 and XIAP). In a phase 2 trial (NCT02503423), tolinapant showed activity against heavily pre-treated patients with TCL. We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the mechanism of synergy between tolinapant and drugs active against PTCL including romidepsin, pralatrexate, and the HMAs; azacytidine and decitabine. Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFα. For combination studies, each drug was tested, in the presence or absence of TNFα. Synergy coefficient was calculated using Excess over Bliss. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, acetylated histones, DNMTs and key apoptosis and necroptosis markers by Western blotting. Results: TCL Lines demonstrated varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed the highest degree of synergism, compared to romidepsin or pralatrexate. Of note, a high degree of synergism was also seen with concentrations 10 folds lower of azacytidine or 100 folds lower of decitabine, added daily (Table). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 and MLKL by western blot analysis, indicating activation of the necroptosis pathway. Conclusions: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a monotherapy and displays a very high degree of synergism in combination with the HMAs, azacytidine and decitabine, also at low concentration, in vitro. The HMAs were also able to activate the necroptosis pathway, providing a possible mechanism for the high degree of synergism displayed when combined with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate phase I trials with the combination of tolinapant and HMAs. Average synergy scores at 48 hrs for drugs used in combination with tolinapant in a CTCL line HH and ALK+ ALCL line SUP-M2. Above 10 indicates synergy.[Table: see text]