Loss of Caspase-8 function in combination with SMAC mimetic treatment sensitizes Head and Neck Squamous Carcinoma to radiation through induction of necroptosis

Abstract

Abbreviations: HNSCC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; CASP8, Caspase-8; MOC1; mouse oral cancer-1; cIAP1/2, cellular inhibitor of apoptosis proteins-1/2; SMAC, second mitochondria-derived activator of caspase; RIP1, receptor-interacting serine/threonine-protein kinase-1; RIP3, receptor-interacting serine/threonine-protein kinase-3; MLKL, mixed lineage kinase domain-like; Z-VAD-FMK, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]fluoromethylketone; XRT, radiation therapy Conflicts of Interest: The authors have no potential conflicts of interest to disclose. Abstract Word Count: 250 Total Number of Figures and Tables: 6 Total Number of Supplementary Figures and Tables: 17 Significance: CASP8 status regulates necroptotic death in HNSCC and this pathway can be exploited therapeutically. AbstractCaspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCC), and mutations of CASP8 are associated with poor overall survival. The distribution of these mutations in HNSCC suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a unique cell death mechanism. Here, we evaluated how CASP8 regulates necroptosis in HSNCC using cell line models and syngeneic mouse xenografts. In vitro, knockdown of CASP8 rendered HNSCCs susceptible to necroptosis induced by a second mitochondria-derived activator of caspase (SMAC) mimetic, Birinapant, when combined with pan-caspase inhibitors Z-VAD-FMK or Emricasan. Strikingly, inhibition of CASP8 function via knockdown or Emricasan treatment was associated with enhanced radiation killing by Birinapant through induction of necroptosis. In a syngeneic mouse model of oral cancer, Birinapant, particularly when combined with radiation delayed tumor growth and enhanced survival under CASP8loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine-protein kinase-3 (RIP3), a key enzyme in the necroptosis pathway was crucial in determining susceptibility to this mode of death. Although an in vitro screen revealed that many HNSCC cell lines were resistant to necroptosis due to low levels of RIP3, patient tumors maintain RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be a relevant therapeutic approach in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.