Abstract 6042: TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer

Abstract

Abstract Cytotoxic and genotoxic therapies are known to induce the prosurvival Inhibitor-kappaB kinase (IKK)-NF-κB/RELA pathway and DNA repair at the G2/M checkpoint, thereby promoting therapeutic resistance, but the coordination of these mechanisms is unclear. Our recent RNAi screen unexpectedly uncovered possible crosstalk between WEE1, a G2/M checkpoint kinase and the TNFα-IKK-NF-κB prosurvival pathway components. siRNAs targeting WEE1 and IKKs both inhibited TNFα-inducible NF-κB activity in a HNSCC reporter line, suggesting a mechanistic linkage. We investigated this potential connection further and studied the effect of WEE1 inhibition using AZD1775 in head and neck squamous cell carcinoma (HNSCC) cells and mouse xenograft models. Increased expression, phosphorylation and cellular localization of IKK-NF-κB and WEE1-CDC2 axis proteins co-occurred in a subset of HNSCC lines, and were inversely co-modulated by TNFα and WEE1 kinase inhibitor AZD1775. WEE1 co-immunoprecipitated with the IKKα/β and RELA protein complex, and AZD1775 or an IKK inhibitor, IKK16, inhibited phosphorylation and kinase activities, further suggesting that these two axes interact with each other. AZD1775 sensitized HNSCC cells to TNFα-induced cytotoxicity, in part by inhibiting IKK-dependent phosphorylation and nuclear localization of NF-κB activation, and expression of target prosurvival protein BCL-2. Combination of AZD1775 with radiation, a known inducer of TNFα- and DNA-mediated cytotoxicity, potently inhibited HPV+/- HNSCC tumor xenografts. Depleting TNFα abolished the anti-tumor activity of combined therapy with AZD1775 and radiation. The findings of our study reveal that TNFα co-modulates a novel interaction between the prosurvival IKK/NF-κB/RELA and WEE1-CDC2 G2/M checkpoint pathway and that components of both the pathways are inhibited by AZD1775. Our mouse xenograft experiments suggest that AZD1775, when combined with radiation, significantly delays the growth of xenograft HNSCC tumors differing in HPV status in a TNFα-dependent manner. The current findings reveal a novel mechanism whereby crosstalk and co-activation of WEE1- and NF-κB-signaling promotes resistance to TNFα-mediated cytotoxicity, which may be targeted using WEE1 inhibitors. Supported by NIDCD projects ZIA-DC-000016, -73 and 74. Citation Format: Ramya Viswanathan, Zhengbo Hu, Jianghong Chen, Xinping Yang, Angel Huynh, Paul Clavijo, Yi An, Yvette Robbins, Christopher Silvin, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6042.