Functional RNAi screening identifies G2/M and kinetochore components as modulators of TNFα/NF-κB pro-survival signaling in head and neck squamous cell carcinoma
posted on 2024-11-08, 09:13authored byEthan MorganEthan Morgan, Anthony D Saleh, Shaleeka Cornelius, Sophie G Carlson, Tiffany Toni, Hui Cheng, Jun Jeon, Ramya Viswanathan, Xinping Yang, Christopher Silvin, Paul E Clavijo, Anastasia L Sowers, James B Mitchell, et al.
Immune and radiation resistance of cancer cells to cytotoxicity mediated by Tumor Necrosis Factor-α (TNFα) is promoted by the transcription factor NF-κB in several cancers, including head and neck squamous cell carcinoma (HNSCC). Genomic alterations that converge on the TNFα/NF-κB signal axis were found in ~40% of HNSCCs by The Cancer Genome Atlas (TCGA). However, identification of therapeutic targets that contribute to aberrant TNFα/NF-κB activation and resistance has been challenging. Here, we conducted a functional RNAi screen to identify regulators of TNFα-induced NF-κB activation and cell viability, using parallel NF-κB β-lactamase reporter and cell viability assays in a HNSCC cell line which harbors expression and genomic alterations typically found in HPV-negative HNSCC. Besides multiple components of canonical TNFα/NF-κB signaling, we identified components of the WNT, NOTCH, and TGFβ pathways that we previously showed contribute to non-canonical activation of NF-κB. Unexpectedly, we also observed that multiple G2/M cell cycle kinases (AURKA, PLK1, WEE1, TTK), and structural kinetochore/microtubule components (NDC80, NUF2), modulate TNFα-induced NF-κB activation and cell viability. Several of these targets inhibit TNF-induced nuclear translocation of RELA, consistent with prior reports linking NF-B activation to G2/M kinases or microtubule assembly. Further investigation of an understudied mitotic kinase, TTK/MPS1, show that it's inhibition or depletion attenuates TNFα-induced RELA nuclear translocation, promoting cell death, DNA damage, polyploidy, and mitotic catastrophe, leading to radiosensitization. Together, our RNAi screening identifies a critical linkage between the G2/M cell cycle checkpoint/kinetochore components and NF-κB activity, and as targets that can sensitize HNSCC cells to TNFα or radiation.