Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB–inducing kinase (NIK)–dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin β receptor (LTβR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)–dependent nuclear translocation of p52, but not of the TNF-α receptor–mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTβR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

核因子κB（NF-κB）转录因子家族的成员对于多种细胞过程至关重要，包括调节先天和适应性免疫应答，细胞增殖和细胞存活。典型的NF-κB复合物被抑制蛋白IκBα保留在细胞质中，而非典型的NF-κB复合物则被p100保留。尽管对通过IκBα激酶复合物激活经典NF-κB信号的方法进行了深入研究，但几乎未发现非经典p100到p52的NF-κB诱导激酶（NIK）依赖性加工的调控因子以及随后的p52核易位。我们发现细胞周期蛋白依赖性激酶12（CDK12）在转录调控非规范性NF-κB通路中的作用。高含量的表型筛选确定了化合物919278是淋巴毒素β受体（LTβR）和肿瘤坏死因子（TNF）受体超家族成员12A（FN14）依赖的p52而不是TNF-α的核易位的特异性抑制剂受体介导的p65核易位。化学蛋白质组学确定CDK12为919278的靶标。919278，CDK抑制剂THZ1或siRNA介导的敲除对CDK12的抑制导致相似的整体转录变化，并阻止了LTβR和FN14依赖性表达通过减少RNA聚合酶II的羧基末端结构域的磷酸化，MAP3K14（编码NIK）以及NIK积累。通过将表型筛选与化学蛋白质组学结合，我们确定了非规范性NF-κB途径的激活途径，该途径可作为自身免疫性和癌症的治疗靶标。