Tolinapant (ASTX660) is an orally bioavailable cIAP1/2 and XIAP antagonist that induces expression of immunogenic cell death (ICD) markers in sensitive HNSCC cell lines in vitro.

METHODS: Four human HNSCC cell lines (3 HPV-, 1 HPV+) were treated with IFN-γ (positive control, 10 ng/mL), TNFα (20 ng/mL), Tolinapant (ASTX660) (500 nM, 1 μM), and ASTX660 (250 nM, 500 nM, 1 μM) + TNFα for 48 hours, and then the intracellular APM components were analyzed by flow cytometry.
RESULTS In all four cell lines, HLA-A, B, and C expression was consistently increased under ASTX660 + TNFα treatment. ASTX660 + TNFα may enhance tumor cell killing. [1]
METHODS: DLD1, HCT116, and SW48 were treated with ASTX660 (0.04, 0.2, 1 μM, 24 hours). After washing and cell lysis, western blot analysis was performed using antibodies specific for the indicated proteins.
RESULTS ASTX660 concentrations as low as 40 nM were sufficient to rapidly reduce cIAP2 (but not cIAP1) levels. [2]

METHODS: Tolinapant (ASTX660)(16 mg/kg, oral), XRT (single dose 8 Gy), or a combination of both treated a MEER syngeneic tumor xenograft mouse model. Tumors, spleens, and DLNs were harvested and analyzed for CD8 lymphocytes by flow cytometry. Tumor subpopulations were digested and T cells were magnetically selected and cocultured with inactivated dendritic cells presenting p15E peptide. Subsequent IFN-γ production was quantified using an ELISpot assay.
RESULTS The number of CD8+ T cells in the spleen was significantly increased in animals treated with ASTX660 alone and in the subpopulation of animals treated with radiation ± ASTX660 (Figure 3(b)). In the draining lymph nodes of animals treated with XRT alone There was a significant increase in the number of CD8 T lymphocytes and an almost significant increase in the number of CD8 T lymphocytes in animals that received the combination treatment. [1]