TNFα是免疫、化疗和放疗诱导的细胞毒性的关键介质，但包括头颈鳞状细胞癌（HNSCC）在内的多种癌症由于激活经典的NFκB pro-survival途径而表现出对TNFα的耐药性。然而，直接靶向这条途径会带来显著的毒性，因此，识别在癌细胞中有助于NFκB活化和TNFα耐药的新机制对此至关重要。在这里，我们证明蛋白酶体相关去泛素化酶-USP14的表达在HNSCC中显著增加，与人乳头状瘤病毒（HPV）-HNSCC中更差的无进展生存率相关。抑制或耗竭USP14可抑制HNSCC细胞的增殖和存活。此外，USP14抑制降低了NFκB活性、NFκB依赖性基因表达和NFκB亚单位RELA的核转位，包括基础和TNFα诱导的活性。在机制上，USP14结合RELA和IκBα，减少IκBα K48-泛素化从而导致IκBα的降解，这是经典NFκB途径的关键抑制因子。此外，我们证明，USP14和UCHL5的抑制剂b-AP15可使HNSCC细胞对TNFα介导的细胞死亡以及放射线诱导的体外细胞死亡变得敏感。最后，b-AP15可延缓HNSCC肿瘤的生长，并在单药治疗和联合放疗的HNSCC肿瘤异种移植模型中增强生存，这可通过TNFα耗竭显著减轻。这些数据为HNSCC中NFκB信号转导的活化提供了新的见解，并证明了作为敏化这些癌症对TNFα和放疗诱导的细胞毒性的新疗法途径，有必要进一步研究针对泛素化途径的小分子抑制剂。© 2023年。这是美国政府的工作，不受版权保护；外国版权保护可能适用。

TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.