Human papillomavirus–negative (HPV⁻) head and neck squamous cell carcinomas (HNSCC) harbor frequent genomic amplification of Fas-associated death domain, with or without concurrent amplification of Baculovirus inhibitor of apoptosis repeat containing (BIRC2/3) genes encoding cellular inhibitor of apoptosis proteins 1/2 (cIAP1/2). Antagonists targeting cIAP1 have been reported to enhance sensitivity of HPV⁻, but not HPV⁺ tumors, to TNF family death ligands (TNF and TRAIL) and radiation.

Experimental Design: We tested a novel dual cIAP/XIAP antagonist ASTX660 in HPV⁺ and HPV⁻ cell lines in combination with death ligands TNFα and TRAIL, and in preclinical xenograft models with radiation, an inducer of death ligands. The dependence of activity on TNF was examined by antibody depletion.

ASTX660 sensitized subsets of HPV⁻ and HPV⁺ HNSCC cell lines to TNFα and TRAIL. These antitumor effects of ASTX660 are the result of both apoptosis and/or necroptosis among HPV⁻ cells, and primarily by apoptosis (caspase 3 and caspase 8 cleavage) in HPV⁺ cells. ASTX660 enhanced restoration of protein expression and inhibitory activity of proapoptotic tumor suppressor TP53 in HPV⁺ HNSCC. Furthermore, ASTX660 combined with radiotherapy, an inducer of death ligands, significantly delayed growth of both HPV⁻ and HPV⁺ human tumor xenografts, an effect attenuated by anti-TNFα pretreatment blockade.

IAP1/XIAP antagonist, ASTX660, sensitizes HPV⁺ HNSCC to TNFα via a mechanism involving restoration of TP53. These findings serve to motivate further studies of dual cIAP/XIAP antagonists and future clinical trials combining these antagonists with radiotherapy to treat both HPV⁺ and HPV⁻ HNSCC.